Polypeptides Selective for avB3 Integrin, Including Pegylated Polypeptides, And Pharmaceutical Uses Thereof

ABSTRACT

The invention provides disintegrin variants, and more specifically to disintegrin variants which are selective αvβ3 integrin antagonists for treatment and prevention of αvβ3 integrin-associated diseases. The present invention also relates to pegylated proteins, including proteins comprising a disintegrin variant having an RGD motif variant  48 ARLDDL 53 . The invention also relates to the use of the polypeptides of the invention for the treatment and prevention of αvβ3 integrin-associated diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority of U.S. patent application Ser. No. 12/004,045 filed Dec. 20, 2007 which claims priority of U.S. provisional patent application Ser. No. 60/871,854 filed Dec. 26, 2006, the contents of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to disintegrin variants, and more specifically to disintegrin variants as selective αvβ3 integrin antagonists for treatment and prevention of αvβ3 integrin-associated diseases. The present invention also relates to pegylated proteins comprising a disintegrin variant having an RGD motif variant ⁴⁸ARLDDL⁵³. The invention also relates to the use of the polypeptides of the invention for the treatment and prevention of αvβ3 integrin-associated diseases.

BACKGROUND OF THE INVENTION

Bone is a complex tissue composed of several cell types which are continuously undergoing a process of renewal and repair termed “bone remodeling.” The two major cell types responsible for bone remodeling are osteoclasts, which resorb bone, and osteoblasts, which form new bone. Bone remodeling has been known to be regulated by several systemic hormones (e.g., parathyroid hormone, 1,25-dihydroxy vitamin D₃, sex hormones, and calcitonin) and local factors (e.g., nitric oxide, prostaglandins, growth factors, and cytokines).

Integrins are heterodimeric matrix receptors that anchor cells to substrates and transmit externally derived signals across the plasma membrane. Integrin αvβ3 is involved in the osteoclast-mediated bone resorption, both in vivo and in vitro. This heterodimer molecule recognizes the amino acid motif Arg-Gly-Asp (RGD) contained in bone matrix proteins such as osteopontin and bone sialoprotein. Integrin αvβ3 is expressed in an osteoclast and its expression is modulated by resorptive steroids and cytokines. Based on blocking experiments, αvβ3 integrin has been identified as a major functional adhesion receptor on osteoclasts. Inhibitors of integrin αvβ3 reduce the capacity of osteoclasts to bind to and resorb bone. Integrin αvβ3 plays a major role in the function of osteoclasts and inhibitors of this integrin are being considered for treating or preventing osteoporosis, osteolytic metastases, and malignancy-induced hypercalcemia.

There are many bone diseases that are related to osteolysis that is mediated by osteoclasts. Osteoporosis is the most common one that is induced when resorption and formation of bone are not coordinated and bone breakdown overrides bone building. Osteoporosis is also caused by other conditions, such as hormonal imbalance, diseases, or medications (e.g., corticosteroids or anti-epileptic agents). Bone is one of the most common sites of metastasis by human breast, prostate, lung and thyroid cancers, as well as other cancers. Osteoporosis may also result from post-menopausal estrogen deficiency. Secondary osteoporosis may be associated with rheumatoid arthritis. Bone metastasis shows a very unique step of osteoclastic bone resorption that is not seen in metastasis of other organs. It is widely accepted that osteolysis that is associated with cancer is essentially mediated by osteoclasts, which seem to be activated and may be indirectly activated through osteoblasts or directly by tumor products. In addition, hypercalcemia (increased blood-calcium concentration) is an important complication of osteolytic bone diseases. It occurs relatively frequently in patients with extensive bone destruction, and is particularly common in breast, lung, renal, ovarian and pancreatic carcinomas and in myeloma.

Disintegrins are a family of low-molecular-weight RGD-containing peptides that bind specifically to integrins αIIbβ3, α5β1 and αvβ3 expressed on platelets and other cells including vascular endothelial cells and some tumor cells. In addition to their potent antiplatelet activity, studies of disintegrins have revealed new uses in the diagnosis of cardiovascular diseases and the design of therapeutic agents in arterial thrombosis, osteoporosis and angiogenesis-related tumor growth and metastasis. Rhodostomin (Rho), a disintegrin derived from the venom of Colloselasma rhodostoma, has been found to inhibit platelet aggregation in vivo and in vitro through the blockade of platelet glycoprotein αIIbβ3. Furthermore, rhodostomin is reported to inhibit the adhesion of breast and prostate carcinoma cells to both unmineralized and mineralized bone extracellular matrices in a dose-dependent manner, without affecting the viability of tumor cells. In addition, rhodostomin inhibits the migration and invasion of breast and prostate carcinoma cells. Rhodostomin has also been shown to inhibit adipogenesis and obesity. However, because rhodostomin non-specifically binds to integrins αIIbβ3, α5β1 and αvβ3, the pharmaceutical uses of rhodostomin may cause serious side effects. For example, when applying rhodostomin in treating carcinomas, the inhibition of platelet aggregation is an undesirable side effect.

The role of αvβ3 integrin in bone diseases has been well documented. See, for example, F. Patrick Ross et al, Nothing but skin and bone, the Journal of Clinical Investigation, Vol. 116, #5, May 2006; S.B. Rodan et al, Integrin function in osteoclasts, Journal of Endocrinology (1997) 154, S47-S56; Steven L. Teitelbaum, Editorial: Osteoporosis and Integrins, the Journal of Clinical Endocrinology and Metabolism, April 2005, 90(4): 2466-2468; Steven L. Teitelbaum, Osteoclasts, integrins, and osteoporosis, Journal of Bone and Mineral Metabolism, (2000) 18: 344-349; Ichiro Nakamura et al, Involvement of αvβ3 integrins in osteoclast function, Journal of Bone and Mineral Metabolism, (2007) 25: 337-344; Le T. Duong et at, The role of integrins in osteoclast function, Journal of Bone and Mineral Metabolism, (1999) 17: 1-6; and A Teti et al, The Role of the AlphaVbeta3 Integrin in the Development of Osteolytic Bone Metastases: A Pharmacological Target for Alternative Therapy?, Calcified Tissue International (2002) 71: 293-299.

In addition to bone diseases, αvβ3 integrin plays an important role in angiogenesis and tumor growth in conditions not related to bone diseases.

Thus, it may be desirable to create polypeptides selective for αvβ3 integrin with improved stability and lasting effects. These polypeptides will be potentially suitable to treat diseases and conditions involving αvβ3 integrin, including but not limited to various bone diseases, cancer, and diseases involving angiogenesis.

It would also be advantageous to create pegylated polypeptides selective for αvβ3 integrin to prolong the duration and decrease the antigenicity of a protein drug.

SUMMARY OF THE INVENTION

In accordance with the invention, one aspect of the invention is a polypeptide that is selective for αvβ3 integrin. The polypeptide exhibits reduced binding to αIIbβ3 and/or α5β1 integrin compared to a wild-type disintegrin. The polypeptide is encoded by a modified disintegrin nucleotide sequence that encodes a modified amino acid sequence, with reduced αIIbβ3 and/or α5β1 integrin binding activity.

The polypeptide may be pegylated or conjugated with albumin or conjugated with a tissue target molecule. More specifically, the invention provides pegylated polypeptides, including but not limited to PEG-ARLDDL which is a pegylated ARLDDL protein.

The disintegrin nucleotide sequence may be derived from snake venom. The disintegrin may be chosen from rhodostomin, albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, flavoridin, flavostatin, halysin, halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, and viridin.

Another aspect of the invention is a polypeptide that is a variant of rhodostomin, in which the rhodostomin comprises the amino acid sequence of SEQ ID NO: 1.

Another aspect of the invention is a polypeptide that comprises an amino acid chosen from SEQ ID NOs: 30-42.

Another aspect of the invention is a polypeptide comprising the amino acid sequence of SEQ ID NO: 29, which further comprises, one, two, three of four amino acid substitutions in positions corresponding to amino acids 48, 50, 52, or 53 of SEQ ID NO: 1.

Another aspect of the invention is a polypeptide comprising amino acid substitutions chosen from Ala at position 48, Leu, Ile, and His at position 50, Asp, Met, and Asn at position 52, and Val, Leu, and Met at position 53 of SEQ ID NO: 1.

Another aspect of the invention is a polypeptide that is an encoded nucleotide sequence chosen from SEQ ID NOs: 43-56.

Another aspect of the invention is a polypeptide that exhibits at least about a 5, 50, or 100-fold decrease in affinity to αIIbβ3 and/or α5β1 as compared to rhodostomin. In one embodiment of the invention, the polypeptide exhibits at least about a 200-fold decrease in affinity to αIIbβ3 and/or α5β1 integrin as compared to rhodostomin. In another embodiment of the invention, the polypeptide exhibits at least about a 1000 or 2000-fold decrease in affinity to αIIbβ3 and/or α5β1 integrin as compared to rhodostomin. In another embodiment of the invention, the polypeptide exhibits at least about 5, 50, 100, 1000, or 2000-fold decrease in affinity to platelet as compared to rhodostomin. In still another embodiment of the invention, the polypeptide exhibits a substantially reduced activity in prolongation of blood clotting time as compared to rhodostomin and or a wild-type disintegrin.

Yet another aspect of the invention is a physiologically acceptable composition comprising a polypeptide of the invention, and a pharmaceutically acceptable carrier.

Another aspect of the invention is a polypeptide comprising an amino acid sequence chosen from SEQ ID NOs: 57-69.

Yet another aspect of the invention is a method of using a disintegrin variant for treatment and/or prevention of an αvβ3 integrin-associated disease in a mammal including a human. The method includes the step of administering to the mammal in need thereof a therapeutically effective amount of a disintegrin variant. The polypeptide employed in the method may be pegylated or conjugated with albumin or conjugated with a tissue target molecule.

In one aspect of the invention, the disintegrin may be derived from a snake venom, and may be chosen from one of rhodostomin, albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, flavoridin, flavostatin, halysin, halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, and viridin.

In one aspect of the invention, the disintegrin is rhodostomin.

In another aspect of the invention, the rhodostomin comprises a variant of rhodostomin comprising the amino acid sequence of SEQ ID NO: 1.

In another aspect, the rhodostomin comprises an RGD motif variant comprising an amino acid chosen from SEQ ID NOs: 30-42.

In another aspect, the rhodostomin comprises an amino acid chosen from SEQ ID NOs: 57-69.

In one aspect of the invention, the αvβ3 integrin-associated disease includes, but is not limited to, osteoporosis, bone tumor or cancer growth and symptoms related thereto, angiogenesis-related tumor growth and metastasis, tumor metastasis in bone, malignancy-induced hypercalcemia, angiogenesis-related eye diseases, Paget's disease, rheumatic arthritis and osteoarthritis.

In another aspect of the invention, a polypeptide of the invention is used for treatment and/or prevention of an angiogenesis-related eye disease, which includes, but is not limited to, age-related macular degeneration, diabetic retinopathy, corneal neovascularizing diseases, ischaemia-induced neovascularizing retinopathy, high myopia, and retinopathy of prematurity.

In still another aspect of the invention, a polypeptide of the invention is used for treatment and/or prevention of osteoporosis. The osteoporosis is may be associated with a pathological condition chosen from post-menopausal estrogen deficiency, secondary osteoporosis, rheumatoid arthritis, ovariectomy, Paget's disease, bone cancer, bone tumor, osteoarthritis, increased osteoclast formation, and increased osteoclast activity. Furthermore, the osteoporosis includes, but is not limited to, an ovariectomy-induced or post-menopausal osteoporosis or bone loss.

Yet another aspect of the invention is a method of using a polypeptide of the invention for treatment and/or prevention of an ovariectomy-induced physiological change in a mammal including a human.

Yet another aspect of the invention is a method of using a disintegrin variant for inhibition and/or prevention of tumor cell growth in bone and symptoms related thereto in a mammal including a human.

Yet another aspect of the invention is a method for making a polypeptide of the invention, the method comprising the steps of: (a) transfecting a host cell with a polynucleotide encoding said polypeptide (b) growing said host cell in a culture medium; and (c) isolating said polypeptide. The method of the invention may further comprise growing host cells in a culture medium free of amino acids; and collecting supernatant to obtain said polypeptide. The method may further comprise adding methanol to the culture medium to induce polypeptide expression in the host cells. The method may further comprise the step of performing column chromatography to obtain said polypeptide. In one embodiment, the method may further comprise the step of performing HPLC to obtain the isolated polypeptide. The method may further comprise pegylating said polypeptide or a fusion of a tissue targeting molecule to the said polypeptide.

Another aspect of the invention is a polynucleotide encoding a polypeptide selective for αvβ3 integrin, wherein the polypeptide may be a variant of a disintegrin isolated from snake venom.

In another aspect of the invention, the disintegrin is chosen from rhodostomin, albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, flavoridin, flavostatin, halysin, halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, and viridin.

In another aspect of the invention, the disintegrin comprises rhodostomin.

In another aspect of the invention, the disintegrin comprises a variant of rhodostomin comprising the amino acid sequence of SEQ ID NO: 1.

In another aspect of the invention, the polypeptide comprises an RGD motif variant having an amino acid sequence chosen from SEQ ID NOs: 30-42.

In another aspect of the invention, the polynucleotide comprises a sequence chosen from SEQ ID NOs: 43-56 and 78-135.

In another aspect of the invention, the polynucleotide may encode the polypeptides with one, two, three of four amino acid substitutions in positions corresponding to amino acids 48, 50, 52, or 53 of SEQ ID NO: 1.

Another aspect of the invention is a polynucleotide that hybridizes under stringent conditions to a polynucleotide of the invention.

Another aspect of the invention is a polypeptide encoded by a polynucleotide that hybridizes to a polynucleotide sequence of the invention.

These and other aspects will become apparent from the following description of the various embodiments taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.

The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention.

Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is a graph showing less impact of RD and HSA-RD and ARLDDL than rhodostomin protein on bleeding time in mice.

FIG. 2A are photographs of trabecular bones showing inhibition of ovariectomy-induced trabecular bone loss in rats treated with PGP protein. Bar=1 mm.

FIG. 2B are photographs of Tartrate-resistant acid phosphatase (TRAP) stained trabecular bones showing inhibition of ovariectomy-induced increase in osteoclast number in rats treated with PGP protein. Bar=100 mm.

FIG. 3 is a graph showing inhibition of ovariectomy-induced osteoclast activation in rats treated with RD protein or alendronate (drug treatment), rebound of the osteoclast marker concentration during the period of drug withdrawal, and reversal of ovariectomy-induced increase in osteoclast activity during re-administration with RD or alendronate.

FIGS. 4A-4D are photographs of safranin-O and haematoxylin stained knee joint showing inhibition of chondrocyte layer destruction in arthritic rats treated with RD protein. Arrows indicate the chondrocyte layer.

FIGS. 5A-5C are graphs showing inhibition of blood cytokine elevation in osteoarthritis rat treated with RD protein.

FIGS. 6A-6C are graphs showing inhibition of blood cytokine elevation in osteoarthritis mice treated with RD protein.

FIG. 7 is a graph showing inhibition of PC-3 bone tumor growth in SCID mice treated with RD protein, but not with alendronate.

FIG. 8 is a graph showing inhibition of tumor-induced decrease in body weight in SCID mice treated with RD protein.

FIG. 9A are photographs showing a visible spherical tumor growth in the proximal tibia of each leg after the intratibial injection of PC-3 cells (shown by arrows in the middle panel) and an inhibition of the bone tumor growth in SCID mice treated with RD protein.

FIG. 9B are radiographs of tibia bones showing inhibition of PC-3 tumor cells-induced osteolytic bone lesion in SCID mice treated with RD protein.

FIG. 9C is a graph of the quantitation of the data in FIG. 9B showing inhibition of PC-3 tumor-induced osteolysis in SCID mice treated with RD protein.

FIG. 9D is a graph showing inhibition of PC-3 tumor-induced increase in C-terminal telopeptides of type-I collagen in SCID mice treated with RD protein.

FIG. 9E is a graph showing inhibition of PC-3 tumor-induced increase in the serum calcium concentration in SCID mice treated with RD protein or alendronate.

FIG. 10A is a graph showing inhibition of MDA-MB-231 bone tumor growth in nude mice treated with RD protein.

FIG. 10B is a graph showing inhibition of MDA-MB-231 bone tumor-induced increase in C-terminal telopeptides of type-I collagen in nude mice treated with RD protein.

FIG. 100 is a graph showing inhibition of MDA-MB-231 bone tumor-induced hypercalcemia in nude mice treated with RD protein.

FIG. 10D is a graph showing no change in white blood cell counts in nude mice injected with MDA-MB-231 cells and treated with RD protein.

FIG. 10E is a graph showing no change in red blood cell counts in nude mice injected with MDA-MB-231 cells and treated with RD protein.

FIG. 10F is a graph showing no change in the platelet counts in nude mice injected with MDA-MB-231 cells and treated with RD protein.

FIG. 11A are photographs showing reduced blood vessel density in MATRIGEL™ plugs from C57BL/6 mice treated with RD or RD-albumin (HSA-RD) protein in comparison with untreated control mice.

FIG. 11B is a graph showing reduced hemoglobin content in MATRIGEL™ plugs from C57BL/6 mice treated with RD (every day-RD/1d or every other day-RD/2d) or RD-albumin (HSA-RD-every other day) protein in comparison with untreated control mice.

FIG. 12A are photographs showing angiogenesis in a mouse model of retinopathy of prematurity (ROP), and reduced angiogenesis in a ROP mouse treated with RD protein (ROP+RD). Arrows indicate blood vessel profiles (BVPs).

FIG. 12B is a graph showing reduced BVPs in a mouse model of retinopathy of prematurity (ROP) treated with RD protein.

FIG. 13A is a graph showing inhibition of ovariectomy-induced osteoclast activation in mice treated with RD or RD-albumin protein.

FIG. 13B is a graph showing inhibition of ovariectomy-induced alkaline phosphatase (ALP) inactivation in mice treated with RD or RD-albumin protein.

FIG. 13C is a graph showing inhibition of ovariectomy-induced decrease in BMD.

FIG. 13D is a graph showing inhibition of ovariectomy-induced decrease in BMC.

FIGS. 14A-D show amino acid sequences SEQ ID NOs: 1, and 57-69 of rhodostomin variants.

FIGS. 15A-C show nucleotide sequences SEQ ID NOs: 43-56 of rhodostomin variants.

FIGS. 16A-H show amino acid and nucleotide sequences SEQ ID NOs: 78-135 of disintegrin variants.

FIG. 17A shows photographs showing reduced blood vessel density in MATRIGEL™ plugs from mice treated with PEG-ARLDDL in comparison with untreated control mice.

FIG. 17B is a bar chart that shows that mice treated with PEG-ARLDDL had significantly lower hemoglobin content in comparison with untreated control mice.

FIG. 18 shows photographs of tumors excised from the control mice; mice treated with 2 mg/kg/week PEG-ARLDDL; and mice treated with 10 mg/kg/week PEG-ARLDDL.

FIG. 19 is a bar chart that shows that PEG-ARLDDL significantly inhibited tumor growth in a dose-dependent manner.

FIG. 20 is a graph that shows that PEG-ARLDDL administered once a week had a stronger inhibitory effect on tumor growth as compared to PEG-ARLDDL administered once every two weeks.

FIG. 21 shows photographs of tumors excised from the control mice; mice treated with 10 mg/kg/week PEG-ARLDDL administered once a week; and mice treated with 10 mg/kg/week PEG-ARLDDL administered twice a week.

FIG. 22 a bar chart that shows that PEG-ARLDDL administered once a week had a stronger inhibitory effect on tumor growth as compared to PEG-ARLDDL administered once every two weeks.

FIG. 23 is a graph that shows that PEG-ARLDDL significantly enhanced the cytotoxic activity of daunomycin.

FIG. 24 shows photographs of tumors excised from the control mice; mice treated with daunomycin alone; mice treated with PEG-ARLDDL alone; and mice treated with a combination of daunomycin and PEG-ARLDDL.

FIG. 25 is a bar chart that shows that PEG-ARLDDL significantly enhanced the cytotoxic activity of daunomycin.

FIG. 26 shows photographs of U87 human glioma cells that demonstrate that PEG-ARLDDL significantly inhibited osteopontin-induced glioma invasion in hypoxia.

FIG. 27 is a bar chart that demonstrates that PEG-ARLDDL significantly inhibited osteopontin-induced glioma invasion in hypoxia.

FIG. 28 shows photographs of a control mouse and its excised tumor and a mouse treated with PEG-ARLDDL and its excised tumor.

FIG. 29 is a chart that shows that PEG-ARLDDL significantly inhibited U87 human glioma tumor growth in mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the invention are now described in detail. As used in the description and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description and throughout the claims that follow, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. Additionally, some terms used in this specification are more specifically defined below. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.

Reference will now be made in detail to the present embodiments (exemplary embodiments) included in the invention, examples of which are illustrated in the accompanying drawings.

Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. The invention is not limited to various embodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.

“Around,” “about” or “approximately” shall generally mean within 20 percent, within 10 percent, within 5, 4, 3, 2, or 1 percent of a given value or range. Numerical quantities given are approximate, meaning that the term “around,” “about” or “approximately” can be inferred if not expressly stated.

The terms “polynucleotide,” “nucleotide,” “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “polynucleotide sequence,” and “nucleotide sequence” are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides can comprise deoxyribonucleotides, ribonucleotides, and/or their analogs or derivatives. The term includes variants. Variants may include insertions, additions, deletions, or substitutions. Nucleotide sequences are listed in the 5′ to 3′ direction.

The terms “polypeptide,” “peptide,” and “protein,” are used interchangeably to refer to a polymeric form of amino acids of any length, which can include naturally-occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or designer amino acids, amino acid analogs, peptidomimetics, and depsipeptides, and polypeptides having modified, cyclic, bicyclic, depsicyclic, or depsibicyclic peptide backbones. The term includes single chain protein as well as multimers. The term also includes proteins conjugated to a label such as FITC, biotin, and radioisotopes, including, but not limited to ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, 99mTc, 111In, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³⁷Cs, ¹⁸⁶Re, ²¹¹At, ²¹²Bi, ²¹³Bi, ²²³Ra, ²⁴¹Am, and ²⁴⁴Cm; enzymes having detectable products (for example, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, and the like); fluorescers and fluorescent labels, fluorescence emitting metals, for example, ¹⁵²Eu, or others of the lanthanide series, electrochemiluniescent compounds, chemiluminescent compounds, for example, luminol, isoluminol, or acridinium salts; specific binding molecules, for example, magnetic particles, microspheres, nanospheres, and the like. The term also includes peptides conjugated to therapeutic agents.

The terms also include fusion proteins, including, but not limited to, glutathione S-transferase (GST) fusion proteins, fusion proteins with a heterologous amino acid sequence such as bioluminescent proteins, for example, luciferin, or aequorin (green fluorescent protein), with heterologous and homologous leader sequences, fusion proteins with or without N-terminal methionine residues, pegylated proteins, and immunologically tagged, or his-tagged proteins. Such fusion proteins also include fusions to epitopes. Such fusion proteins can comprise multimers of the peptides of the invention, e.g. homodimers or homomultimers, and heterodimers and heteromultimers. The term also includes peptide aptamers.

The term “hybridizes specifically,” in the context of a polynucleotide, refers to hybridization under stringent conditions. Conditions that increase stringency of both DNA/DNA and DNA/RNA hybridization reactions are widely known and published in the art. Examples of stringent hybridization conditions include hybridization in 4× sodium chloride/sodium citrate (SSC), at about 65-70° C., or hybridization in 4×SSC plus 50% formamide at about 42-50° C., followed by one or more washes in 1× SSC, at about 65-70° C.

The term “ligand” refers to a molecule that binds to another molecule, including a receptor.

A “host cell” is an individual cell or cell culture which can be or has been a recipient of any recombinant vector(s) or polynucleotide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell which comprises a recombinant vector of the invention may be called a “recombinant host cell.”

“Treatment,” covers any administration or application of remedies for disease in a mammal, including a human, and includes inhibiting the disease, arresting its development, or relieving the disease, for example, by causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process. The term includes obtaining a desired pharmacologic and/or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal, including a human. The effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse affect attributable to the disorder. Thus, the invention provides both treatment and prophylaxis. It includes (1) preventing the disorder from occurring or recurring in a subject who may be predisposed to the disorder but is not yet symptomatic, (2) inhibiting the disorder, such as arresting its development, (3) stopping or terminating the disorder or at least its associated symptoms, so that the host no longer suffers from the disorder or its symptoms, such as causing regression of the disorder or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the disorder, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, and/or tumor size.

The phrase “cancer associated with a highly expressed osteopontin (OPN)” refers to a cancer wherein the expression level of OPN is higher than the expression level of OPN in peripheral normal tissues.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material, formulation auxiliary, or excipient of any conventional type. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

A “composition” refers to a mixture that usually contains a carrier, such as a pharmaceutically acceptable carrier or excipient that is conventional in the art and which is suitable for administration into a subject for therapeutic, diagnostic, or prophylactic purposes. It may include a cell culture in which the polypeptide or polynucleotide is present in the cells or in the culture medium. For example, compositions for oral administration can form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses, or powders.

“Disease” refers to any condition, infection, disorder, or syndrome that requires medical intervention or for which medical intervention is desirable. Such medical intervention can include treatment, diagnosis, and/or prevention.

Peptides

The peptides of the invention can be expressed using methods known in the art. Cell-based methods and cell-free methods are suitable for producing peptides of the invention. Cell-based methods generally involve introducing a nucleic acid construct into a host cell in vitro and culturing the host cell under conditions suitable for expression, then harvesting the peptide, either from the culture medium or from the host cell, (for example, by disrupting the host cell), or both. The invention also provides methods of producing a peptide using cell-free in vitro transcription/translation methods, which are well known in the art.

Suitable host cells include prokaryotic or eukaryotic cells, including, for example, bacterial, yeast, fungal, plant, insect, and mammalian cells.

Typically, a heterologous peptide, whether modified or unmodified, may be expressed on its own, as described above, or as a fusion protein, and may include not only secretion signals, but also a secretory leader sequence. A secretory leader sequence of the invention may direct certain proteins to the endoplasmic reticulum (ER). The ER separates the membrane-bound proteins from other proteins. Once localized to the ER, proteins can be further directed to the Golgi apparatus for distribution to vesicles, including secretory vesicles, the plasma membrane, lysosomes, and other organelles.

Additionally, peptide moieties and/or purification tags may be added to the peptides. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability, and to facilitate purification, among other reasons, are familiar and routine techniques in the art. Suitable purification tags include, for example, V5, polyhistidines, avidin, and biotin. Conjugation of peptides to compounds such as biotin can be accomplished using techniques well known in the art. (Hermanson ed. (1996) Bioconjugate Techniques; Academic Press). Peptides can also be conjugated with radioisotopes, toxins, enzymes, fluorescent labels, colloidal gold, nucleic acids, vinorelbine, and doxorubicin using techniques known in the art. (Hermanson ed. (1996) Bioconjugate Techniques; Academic Press; Stefano et al. (2006).

Fusion partners suitable for use in the invention include, for example, fetuin, human serum albumin, Fc, and/or one or more of their fragments. Conjugated proteins, such as polyethylene glycol conjugates, are also provided.

The peptides of the invention can also be chemically synthesized using techniques known in the art (e.g., see Hunkapiller et al., Nature, 310:105 111 (1984); Grant ed. (1992) Synthetic Peptides, A Users Guide, W.H. Freeman and Co.; U.S. Pat. No. 6,974,884)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer or through the use of solid-phase methods known in the art.

Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levo rotary).

The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. In one embodiment, high performance liquid chromatography (“HPLC”) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.

A peptide or peptidomimetic of the invention can be modified with or covalently coupled to one or more of a variety of hydrophilic polymers to increase solubility and circulation half-life of the peptide. Suitable nonproteinaceous hydrophilic polymers for coupling to a peptide include, but are not limited to, polyalkylethers as exemplified by polyethylene glycol and polypropylene glycol, polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran, and dextran derivatives. Generally, such hydrophilic polymers have an average molecular weight ranging from about 500 to about 100,000 daltons, from about 2,000 to about 40,000 daltons, or from about 5,000 to about 20,000 daltons. The peptide can be derivatized with or coupled to such polymers using any of the methods set forth in Zallipsky, S. (1995) Bioconjugate Chem., 6:150-165; Monfardini, C., et al. (1995) Bioconjugate Chem. 6:62-69; U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; 4,179,337, or WO 95/34326.

In some embodiments, a peptide of the invention is provided in formulation with pharmaceutically acceptable carriers, excipients, and diluents, of which a wide variety are known in the art. These pharmaceutical carriers, excipients, and diluents include those listed in the USP pharmaceutical excipients listing. USP and NF Excipients, Listed by Categories, p. 2404-2406, USP 24 NF 19, United States Pharmacopeial Convention Inc., Rockville, Md. (ISBN 1-889788-03-1). Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the formulation. Topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents can be added as necessary. Percutaneous penetration enhancers such as Azone can also be included.

In pharmaceutical dosage forms, the compositions of the invention can be administered in the form of their pharmaceutically acceptable salts, or they can also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The subject compositions are formulated in accordance to the mode of potential administration.

Methods of Treatment

An αvβ3 integrin-associated disease, includes, but is not limited to, osteoporosis, cancer, bone tumor or cancer growth and symptoms related thereto, angiogenesis-related tumor growth and metastasis, tumor metastasis in bone, malignancy-induced hypercalcemia, angiogenesis-related eye diseases, Paget's disease, rheumatic arthritis and osteoarthritis.

Peptides of the invention may be administered to a subject in need of treatment by systemic injection, such as by intravenous injection; or by injection or application to the relevant site, such as by direct injection, or direct application to the site when the site is exposed in surgery; or by topical application, such as if the disorder is on the skin, for example.

Peptides of the invention can be used as monotherapy. Alternatively, the peptides of the invention can be used in combination with standard regimens to treat αvβ3 integrin associated diseases.

For example, the peptides of the invention can be used in a combinational therapy with a therapeutically effective amount of one or more other pharmaceutical agents.

Preferably, another pharmaceutical agent is selected from the group consisting of an anti-cancer agent, an anti-inflammatory agent, an immune-modulating agent and an anti-osteoporosis agent.

Preferably, an anti-cancer agent is selected from the group consisting of an anti-angiogenic agent, a cytotoxic agent and an anti-neoplastic agent.

The other pharmaceutical agent(s) can be administered prior to, together with, or after administration of the peptides of the invention.

In some embodiments, the combinational therapy with other pharmaceutical agents provides synergistic effects, including but not limited to enhancing the therapeutic efficacy of the other agents; allowing using lower doses of the other agents, thereby decreasing their side effects and reducing resistance to the other agents; and lowering the likelihood and/or the rate of cancer recurrence.

In some embodiments, the polypeptides of the invention are particularly effective against cancers which are associated with a highly expressed osteopontin. In preferred embodiments, the polypeptides of the invention can inhibit osteopontin-induced tumor invasion.

Administration of the agents can be achieved in various ways, including oral, buccal, nasal, rectal, parenteral, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, and intrathecal administration, etc., or otherwise by implantation or inhalation. Thus, the subject compositions can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. The following methods and excipients are merely exemplary and are in no way limiting.

Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle can contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. The composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.

Peptides of the invention can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives. Other formulations for oral or parenteral delivery can also be used, as conventional in the art.

“Disintegrins” refer to a family of polypeptides that may be purified from snake venoms, which contain the arginine-glycine-aspartic acid (RGD) sequence. Without being bound by any theory or mechanism, it is believed that the RGD tripeptide binds with high affinity to integrins to block the interaction of integrins with RGD-containing proteins. Disintegrins thus block adhesive functions and act as platelet aggregation inhibitors.

The abbreviation “Rho” means “rhodostomin,” which is a disintegrin derived from the venom of Colloselasma rhodostoma. Rhodostomin non-specifically binds to integrins αIIbβ3, α5β1 and αvβ3, and prolongs blood clotting time by inhibiting platelet aggregation through the blockade of platelet glycoprotein αIIbβ3.

The “disintegrin variant” or “rhodostomin variant” refers to a functionally active protein, or a polypeptide or any derivatives thereof, that comprises an amino acid sequence derived or modified or mutated from a wild-type disintegrin such as rhodostomin. A functionally active disintegrin/rhodostomin variant can specifically bind to and inhibit integrin αvβ3 activity. The disintegrin or rhodostomin variant of the present invention can be constructed by any method suitable to the aims of the present invention, and in one embodiment by a site-directed mutagenesis method, and in another embodiment by a polymerase chain reaction method. Variants may include insertions, additions, deletions, or substitutions compared with the subject peptides. Variants of polypeptide sequences include biologically active polymorphic variants.

Peptides of the invention can include naturally-occurring and non-naturally occurring amino acids. Peptides can comprise D-amino acids, a combination of D- and L-amino acids, and various “designer” or “synthetic” amino acids (for example, β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids, etc.) to convey special properties. Additionally, peptides can be cyclic. Peptides can include non-classical amino acids in order to introduce particular conformational motifs. Any known non-classical amino acid can be used. Amino acid analogs and peptidomimetics can be incorporated into a peptide to induce or favor specific secondary structures, including, but not limited to, LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducing dipeptide analog; β-sheet inducing analogs; β-turn inducing analogs; α-helix inducing analogs; γ-turn inducing analogs; Gly-Ala turn analogs; amide bond isostere; or tretrazol, and the like.

A desamino or descarboxy residue can be incorporated at the terminal ends of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict conformation. C-terminal functional groups include amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.

The term “IC₅₀,” or “the half maximal inhibitory concentration” refers to the concentration of Rho or its variant that is required for 50% inhibition of its receptor. IC₅₀ is a measure of how much of Rho or its variant is needed to inhibit a biological process by 50%, such as the variant's affinity to its receptor.

The term “therapeutically effective amount” as used refers to an amount which, when administered to a living subject, achieves a desired effect on the living subject. For example, an effective amount of the disintegrin or Rho variant of the invention for administration to the living subject is an amount that prevents and/or treats an integrin αvβ3-mediated disease. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

The term “receptor antagonist” refers to a binding ligand of a receptor that inhibits the function of a receptor by blocking the binding of an agonist to the receptor, or which allows agonist binding, but inhibits the ability of the agonist to activate the receptor.

The term “substantially reduced integrin αIIbβ3 and/or α5β1 receptor-blocking activity” refers to a reduced activity of at least five fold in blocking integrin αIIbβ3 and/or α5β1 receptor compared to wild type rhodostomin or other disintegrins. For example, to calculate the reduction in αIIbβ3 and/or α5β1 receptor-blocking activity, the IC₅₀ of a rhodostomin variant for inhibition of integrin αIIbβ3 and/or α5β1 binding to a matrix protein, such as fibrinogen, is compared to of the IC₅₀ of Rho.

The term “RGD motif variant” refers to a peptide comprising a modification in the amino acid sequence that spans the RGD sequence of a corresponding wild-type sequence, such as the sequence comprising RGD in Rhodostomin. Examples of “RGD motif variants” include ⁴⁸ARGDDP⁵³, ⁴⁸PRLDMP⁵³, ⁴⁸PRIDMP⁵³, and ⁴⁸ARLDDL⁵³.

The term “RD” refers to a rhodostomin variant having a RGD motif variant ⁴⁸PRLDMP⁵³.

The term “PGP” refers to a rhodostomin variant having a RGD motif variant ⁴⁸PRGDGP⁵³.

The term “ARLDDL” refers to a rhodostomin variant having a RGD motif variant ⁴⁸ARLDDL⁵³.

The term “inhibitory selectivity for integrin αvβ3 relative to αIIbβ3 and/or α5β1 receptors” refers to a variant's binding selectivity toward integrin αvβ3 over αIIbβ3 and/or α5β1 receptors, which is expressed as a ratio of the IC₅₀ of the variant for inhibition of αIIbβ3 and/or α5β1 receptors over that for inhibition of αvβ3 receptor.

The term “substantially reduced activity in prolongation of blood bleeding time” refers to a reduced ability to inhibit blood clotting in a statistically significant manner as measured by the bleeding time experiment described in the specification.

The term “PEG-ARLDDL” refers to a pegylated ARLDDL protein.

The terms “albumin-ARLDDL” or “HSA-ARLDDL” refers to a human albumin-conjugated product of RD protein.

Overview of the Invention

The present invention is related to disintegrin variants that are selective αvβ3 integrin antagonists. Disintegrin variants such as RD-related compounds potently inhibit osteoclast differentiation in vitro. They also inhibit osteoclast resorbing activity and ovariectomy-induced increase in osteoclast formation in animal studies. In addition, RD inhibits the tumor growth of human prostate and breast cancer cells in bone. Malignancy-induced hypercalcemia was also effectively blocked by RD-related proteins. Paget's disease (also known as osteitis deformans) is a chronic bone disorder that typically results in enlarged and deformed bones due to irregular breakdown and formation of bone tissues. Bisphosphonates have been approved for the treatment of Paget's disease. Osteoarthritis is also related to the increase in osteoclast activity. Based on the similar mechanism of action, RD derivatives should also be effective for treatment of these bone disorders. An intravenous injection of RD or PGP at a very large dose at 30 mg/kg did not affect the survival of mice (n=3). In addition, long term administration of PGP (I.V., 0.5 mg/kg/day) for 6 weeks did not affect serum level of creatinine, GOT, and GPT, suggesting lack of side effects on kidney and liver. Therefore, RD and its derivatives are potential drug candidates for treatment of osteoporosis, bone tumor, malignancy-induced hypercalcemia, Paget's disease, rheumatic arthritis, osteoarthritis and angiogenesis-related eye diseases.

Many kinds of snake venom contain proteins that comprise an RGD domain. These RGD domain-containing proteins are called disintegrins. A modification in the sequence spanning the RGD domain results in a very unique polypeptide variant with a reduced binding affinity to other kinds of integrins but an increased selectivity to αvβ3 integrin. The disintegrin variants including rhodostomin variants prove to be potential therapeutic candidates for, among others, osteoporosis, suppression of tumor growth in bone, and angiogenesis-related eye diseases. Moreover, disintegrin variants including rhodostomin variants having a RGD-motif region with at least one amino acid substitution may be valuable tools for developing selective antagonists of αvβ3 integrin.

One aspect of the invention is a polypeptide that has integrin αvβ3 receptor-antagonist activity and reduced integrin αIIbβ3 and/or α5β1 receptor-blocking activity as compared to a wild-type disintegrin. The polypeptide is encoded by a modified disintegrin nucleotide sequence that encodes a modified amino acid sequence, which results in a polypeptide having substantially reduced integrin αIIbβ3 and/or α5β1 receptor-blocking activity. The polypeptide may be pegylated or conjugated with albumin or conjugated with a tissue target molecule.

The disintegrin nucleotide sequence may be derived from snake venom. According to the invention, disintegrins include, but are not limited to, albolabrin (Cryptelytrops albolabris), applagin (Agkistrodon piscivorus piscivorus), basilicin (Crotalus basiliscus), batroxostatin (Bothrops atrox), bitistatin (Bitis arietans), cereberin (Crotalus oreganus cerberus), cerastin (Crotalus cerastes cerastes), crotatroxin (Crotalus atrox), durissin (Crotalus durissus durissus), elegantin (Protobothrops elegans), flavoridin (Trimeresurus flavoviridis), flavostatin (Trimeresurus flavoviridis), halysin (Gloydius blomhoffi), halystatin (Gloydius halys), jararacin (Bothrops jararaca), jarastatin (Bothrops jararaca), kistrin (Calloselasma rhodostoma), lachesin (Lachesis muta muta), lutosin (Crotalus oreganus lutosus), molossin (Crotalus molossus molossus), salmosin (Gloydius blomhoffi brevicaudus), saxatilin (Gloydius halys), tergeminin (Sistrurus catenatus tergeminus), trimestatin (Trimeresurus flavoviridis), trimucrin (Protobothrops mucrosquamatus), trimutase (Protobothrops mucrosquamatus), ussuristatin (Gloydius ussuriensis), virid in (Crotalus viridis).

Another aspect of the invention is an isolated polypeptide that is a variant of rhodostomin, in which the rhodostomin comprises an amino acid sequence defined by SEQ ID NO: 1, and the variant comprises an RGD motif variant.

In one embodiment of the invention, the RGD motif variant may contain an amino acid sequence chosen from SEQ ID NOs: 30-42.

Another aspect of the invention is a polypeptide comprising the amino acid sequence of SEQ ID NO: 29, which further comprises, one, two, three of four amino acid substitutions in positions corresponding to amino acids 48, 50, 52, or 53 of SEQ ID NO: 1.

Another aspect of the invention is a polypeptide comprising amino acid substitutions chosen from Ala at position 48, Leu, Ile, and His at position 50, Asp, Met, and Asn at position 52, and Val, Leu, and Met at position 53 of SEQ ID NO: 1.

Another aspect of the invention is a polypeptide that is encoded with a nucleotide sequence chosen from SEQ ID NOs: 43-56.

For example, and specifically, in another embodiment of the invention, the RGD motif variant may comprise at least one amino acid substitution at a residue corresponding to Gly⁵⁰ or Met⁵² of the wild-type RGD motif set forth by SEQ ID NO: 29. The at least one amino acid substitution occurs at a residue corresponding to Leu⁵⁰ of SEQ ID NO: 36-37, Ile⁵⁰ of SEQ ID NO: 39, His⁵⁰ of SEQ ID NO: 40, Asn⁵² of SEQ ID NO: 41, or Gly⁵² of SEQ ID NO: 42.

In another embodiment of the invention, the RGD motif variant may comprise at least two amino acid substitutions at residues corresponding to Pro⁴⁸ and Met⁵², or Met⁵² and Pro⁵³ of the wild-type RGD motif set forth by SEQ ID NO: 29. The at least two amino acid substitutions may be residues corresponding to Ala⁴⁸ and Asp⁵² of SEQ ID NO: 30, or Asp⁵² and Met⁵³ of SEQ ID NO: 35.

In still another embodiment of the invention, the RGD motif variant may comprise at least three amino acid substitutions at residues corresponding to Pro⁴⁸, Met⁵² and Pro⁵³, or Gly⁵⁰, Met⁵² and Pro⁵³ of the wild-type RGD motif set forth by SEQ ID NO: 29. The at least three amino acid substitutions may be residues corresponding to Ala⁴⁸, Asp⁵² and Val⁵³ of SEQ ID NO: 31, Ala⁴⁸, Asp⁵² and Leu⁵³ of SEQ ID NO: 32, Ala⁴⁸, Asp⁵² and Met⁵³ of SEQ ID NO: 34, Leu⁵⁰, Asp⁵² and Leu⁵³ of SEQ ID NO: 37.

In still another embodiment of the invention, the RGD motif variant may comprise at least four amino acid substitutions at residues corresponding to Pro⁴⁸, Gly⁵⁰, Met⁵² and Pro⁵³ of the wild-type RGD motif set forth by SEQ ID NO: 29. The at least four amino acid substitutions may be residues corresponding to Ala⁴⁸, Leu⁵⁰, Asp⁵² and Leu⁵³ of SEQ ID NO: 38.

Another aspect of the invention is an isolated polypeptide that is encoded by a DNA having a modified rhodostomin nucleotide sequence chosen from SEQ ID NOs: 44-56. The polypeptide exhibits at least about a 5, 50, or 100-fold decrease in affinity to αIIbβ3 and/or α5β1 as compared to rhodostomin. In one embodiment of the invention, the polypeptide exhibits at least about a 200-fold decrease in affinity to αIIbβ3 and/or α5β1 integrin as compared to rhodostomin. In another embodiment of the invention, the polypeptide exhibits at least about a 1000 or 2000-fold decrease in affinity to αIIbβ3 and/or α5β1 integrin as compared to rhodostomin. In another embodiment of the invention, the polypeptide exhibits at least about 5, 50, 100, 1000, or 2000-fold decrease in affinity to platelet as compared to rhodostomin. In still another embodiment of the invention, the polypeptide exhibits a substantially reduced activity in prolongation of blood clotting time as compared to rhodostomin or wild-type disintegrin.

Yet another aspect of the invention is a physiologically acceptable composition comprising a polypeptide of the invention and a pharmaceutically acceptable carrier.

Yet another aspect of the invention is a method of using a disintegrin variant for treatment and/or prevention of an αvβ3 integrin-associated disease in a mammal including a human. The method includes the step of administering to the mammal in need thereof a therapeutically effective amount of an isolated polypeptide, or a pharmaceutically acceptable salt thereof. The polypeptide therein has integrin αvβ3 receptor-antagonist activity and substantially reduced integrin αIIbβ3 and/or integrin α5β1 receptor-blocking activity as compared to a wild-type disintegrin, and thereby results in treatment and/or prevention of the αvβ3 integrin-associated disease in the mammal. The polypeptide is encoded by a modified disintegrin nucleotide sequence that encodes a modified disintegrin amino acid sequence, which results in a polypeptide having substantially reduced integrin αIIbβ3 and/or integrin α5β1 receptor-blocking activity. The polypeptide employed in the method may be pegylated or conjugated with albumin or conjugated with a tissue target molecule.

As described above, the disintegrin nucleotide sequence may be derived from a snake venom, and may be chosen from rhodostomin, albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, flavoridin, flavostatin, halysin, halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, and viridin.

In one embodiment of the invention, the method includes the step of administering to the mammal in need thereof a therapeutically effective amount of an isolated polypeptide, or a pharmaceutically acceptable salt thereof, in which the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, and the variant comprises an RGD motif variant.

In another embodiment of the invention, the method includes the step of administering to the mammal in need thereof a therapeutically effective amount of an isolated polypeptide, or a pharmaceutically acceptable salt thereof, in which the polypeptide is a variant of rhodostomin comprising the amino acid sequence defined by SEQ ID NO: 1, and the variant comprises a RGD motif variant having an amino acid sequence chosen from SEQ ID NOs: 30-42.

In still another embodiment of the invention, the method includes the step of administering to a mammal in need thereof a therapeutically effective amount of an isolated polypeptide, or a pharmaceutically acceptable salt thereof, in which the polypeptide comprises an amino acid sequence chosen from SEQ ID NOs: 57-69.

In one embodiment of the invention, a polypeptide of the invention is used for treatment and/or prevention of an αvβ3 integrin-associated disease, which includes, but is not limited to, osteoporosis, cancer,bone tumor or cancer growth and symptoms related thereto, angiogenesis-related tumor growth and metastasis, tumor metastasis in bone, malignancy-induced hypercalcemia, angiogenesis-related eye diseases, Paget's disease, rheumatic arthritis, and osteoarthritis.

In another embodiment of the invention, a polypeptide of the invention is used for treatment and/or prevention of an angiogenesis-related eye disease, which includes, but is not limited to, age-related macular degeneration, diabetic retinopathy, corneal neovascularizing diseases, ischaemia-induced neovascularizing retinopathy, high myopia, and retinopathy of prematurity.

In still another embodiment of the invention, a polypeptide of the invention is used for treatment and/or prevention of osteoporosis. The osteoporosis is associated with a pathological condition chosen from post-menopausal estrogen deficiency, secondary osteoporosis, rheumatoid arthritis, ovariectomy, Paget's disease, bone cancer, bone tumor, osteoarthritis, increased osteoclast formation, and increased osteoclast activity. Furthermore, the osteoporosis includes, but is not limited to, an ovariectomy-induced osteoporosis or bone loss and post-menopausal osteoporosis or bone loss.

Yet another aspect of the invention is a method of using a disintegrin variant for treatment and/or prevention of physiological changes in a mammal including a human induced by ovariectomy or post-menopausal osteoporosis. The method includes administering to the mammal in need thereof a therapeutically effective amount of an isolated polypeptide, or a pharmaceutically acceptable salt thereof, which has integrin αvβ3 receptor-antagonist activity and substantially reduced integrin αIIbβ3 and/or α5β1 receptor-blocking activity as compared to a wild-type disintegrin, and thereby resulting in treatment and/or prevention of the ovariectomy-induced physiological change in the mammal. The polypeptide is encoded by a modified disintegrin nucleotide sequence that encodes a modified amino acid sequence resulting in said polypeptide having substantially reduced integrin αIIbβ3 and/or α5β1 receptor-blocking activity. The disintegrin nucleotide sequence may be derived from a snake venom, and may be chosen from one of rhodostomin, albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, flavoridin, flavostatin, halysin, halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, and viridin.

The polypeptide variant used for treatment and/prevention of an ovariectomy-induced or post-menopausal physiological change in the mammal may contain an RGD motif variant that comprises an amino acid sequence chosen from SEQ ID NOs: 30-42.

In one embodiment of the invention, the method includes administering to the mammal in need thereof a therapeutically effective amount of an isolated polypeptide, or a pharmaceutically acceptable salt thereof, in which the polypeptide variant comprises an amino acid sequence chosen from SEQ ID NOs: 57-69. In another embodiment, the polypeptide variant is pegylated or conjugated with albumin or conjugated with a tissue target molecule.

Yet another aspect of the invention is a method of using a disintegrin variant for inhibition and/or prevention of tumor cell growth in bone and symptoms related thereto in a mammal including a human. The method includes administering to the mammal in need thereof a therapeutically effective amount of an isolated polypeptide, or a pharmaceutically acceptable salt thereof, which has integrin αvβ3 receptor-antagonist activity and substantially reduced integrin αIIbβ3 and/or α5β1 receptor-blocking activity as compared to a wild-type disintegrin, and thereby resulting in inhibition and/or prevention of tumor cell growth in bone and symptoms related thereto in the mammal. The polypeptide is encoded by a modified disintegrin nucleotide sequence that encodes a modified amino acid sequence and thereby results in said polypeptide having substantially reduced integrin αIIbβ3 and/or α5β1 receptor-blocking activity.

The disintegrin nucleotide sequence may be derived from a snake venom, and may be chosen from rhodostomin, albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, flavoridin, flavostatin, halysin, halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, and viridin.

The pathological symptoms related to tumor cell growth in bone include an increased osteoclast activity, an increased bone resorption, bone lesion, hypercalcemia, a body weight loss, and any combinations thereof. The tumor cell growth in bone includes bone cancer cells and metastasized cancer cells originating from prostate cancer, breast cancer, lung cancer, renal cancer, ovarian cancer, pancreatic cancer, myeloma cancer, hepatoma, and melanoma.

In one embodiment of the invention, the method includes administering to a mammal in need thereof a therapeutically effective amount of an isolated polypeptide, or a pharmaceutically acceptable salt thereof, in which the polypeptide is a variant of rhodostomin, in which rhodostomin comprises the amino acid sequence defined by SEQ ID NO: 1, and the variant comprises an RGD motif variant. The RGD motif variant may comprise an amino acid sequence chosen from SEQ ID NOs: 30-42.

In another embodiment of the invention, the method includes the step of administering to a mammal in need thereof a therapeutically effective amount of an isolated polypeptide, or a pharmaceutically acceptable salt thereof, in which the polypeptide comprises an amino acid sequence chosen from SEQ ID NOs: 57-69. The polypeptide variant may be pegylated or conjugated with albumin or conjugated with a tissue target molecule.

Yet another aspect of the invention is a method for making a polypeptide of the invention, the method comprising the steps as follows: (a) transforming a host cell with a polynucleotide encoding said polypeptide to obtain one or more than one transformant; (b) selecting a transformant having one or more than one copy of said DNA construct inserted into the tansformant; (c) growing the transformant in a culture medium to amplify cell population thereof; (d) harvesting transformant cells; (e) growing the harvested transformant cells in a culture medium free of amino acids; and (g) collecting supernatant to obtain said polypeptide.

The aforementioned step (e) may further comprise the step of adding methanol to the culture medium to induce the polypeptide expression in the transformant cells. In one embodiment, the step (g) may further comprise the step of performing column chromatography to obtain said polypeptide. In a further embodiment, the aforementioned method may further comprise the step of performing HPLC to obtain the purified, isolated polypeptide.

These and other aspects of the present invention are more specifically described below.

Human recombinant RANKL and M-CSF were purchased from R&D Systems (Minneapolis, Minn.). The C-terminal telopeptides of type-I collagen ELISA kit was obtained from Cross Laps (Herlev, Denmark). All other chemicals were obtained from Sigma.

EXAMPLE 1 Construction of DNAs Encoding Rhodostomin and Variants

Rhodostomin was cloned and expressed in the vector pGEX-2KS as a template. The DNA encoding Rho was composed of codons preferentially used in Pichia pastoris. Rho DNA was amplified by the polymerase chain reaction (PCR) with the sense primer 5′-GAATTCGAATTCCATCATCATCATCATCAT CATGGTAAGGAATGTGACTGTTCTT-3′ (Rho-Pic-1; SEQ ID NO: 3) that had Eco R1 recognition and six histidine residues for facilitating purification. The antisense primer is 5′-CCGCGGCCGCGGTCAGTGGTATCTTGGACAGTCAGC-3′ or 5′-CCGCGGCCGCGGTTAGTGGTATCTTGGACAGTCAGC-3′ (Rho-Pic-2; SEQ ID NO: 4) with Sac II recognition and a TCA (or TTA) stop codon. The PCR product was purified and then ligated into the Eco R1 and Sac II sites of the yeast recombination vector, pPICZαA. The recombinant plasmid was used to transform a DH5α strain, and colonies were selected on agar plates with low salt LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar at pH 7.0) and 25 μg/ml antibiotic Zeocin.

Rhodostomin variants were synthesized and amplified by the polymerase chain reaction (PCR) using an overlapping oligonucleotide strategy with primers containing Eco RI and Sac II restriction sites. The nucleotide sequences of various primers used for synthesizing or confirming variants are listed in Table 1. RD-HSA fusion protein was constructed using similar procedures. The cDNA of human serum albumin was purchased from Invitrogen, and the structural gene of albumin was fused at N-terminus of Rho gene with a GSGSGS linker amino acid sequence and with six histidine residues at the N-terminus.

TABLE 1 SEQ ID Primer Sequence NO: RLD-1 Sense GAATCCCAAGACTTGACATGCCAG (SEQ ID NO: 5) RLD-2 Anti- CTGGCATGTCAAGTCTTGGGATTC (SEQ ID sense NO: 6) RHD-1 Sense AGAATCCCAAGACACGACATGCCAGAC (SEQ ID NO: 7) RHD-2 Anti- GTCTGGCATGTCGTGTCTTGGGATTCT (SEQ ID sense NO: 8) RID-1 Sense AGAATCCCAAGAATCGACATGCCAGAC (SEQ ID NO: 9) RID-2 Anti- GTCTGGCATGTCGATTCTTGGGATTCT (SEQ ID sense NO: 10) P48A-1 Sense TGTAGAATCGCTAGAGGTGACATG (SEQ ID NO: 11) P48A-2 Anti- CATGTCACCTCTAGCGATTCTACA (SEQ ID sense NO: 12) ARGD-2 Anti- GTCACCTCTTGCGATTCTACAG (SEQ ID sense NO: 13) RGDNP-1 Sense CAAGAGGTGACAACCCAGACGACAG (SEQ ID NO: 14) RGDNP-2 Anti- CTGTCGTCTGGGTTGTCACCTCTTG (SEQ ID sense NO: 15) RGDDP-1 Sense CAAGAGGTGACGACCCAGACGACAG (SEQ ID NO: 16) RGDDP-2 Anti- CTGTCGTCTGGGTCGTCACCTCTTG (SEQ ID sense NO: 17) RGDGP-1 Sense CAAGAGGTGACGGTCCAGACGACAGATG (SEQ ID NO: 18) RGDGP-2 Anti- CATCTGTCGTCTGGACCGTCACCTCTTG (SEQ ID sense NO: 19) RGDDL-1 Sense CAAGAGGTGACGACCTAGACGACAGATG (SEQ ID NO: 20) RGDDL-2 Anti- CATCTGTCGTCTAGGTCGTCACCTCTTG (SEQ ID sense NO: 21) RGDDM-1 Sense CAAGAGGTGACGACATGGACGACAGATG (SEQ ID NO: 22) RGDDM-2 Anti- CATCTGTCGTCCATGTCGTCACCTCTTG (SEQ ID sense NO: 23) RGDDV-1 Sense CAAGAGGTGACGACGTAGACGACAGATG (SEQ ID NO: 24) RGDDV-2 Anti- CATCTGTCGTCTACGTCGTCACCTCTTG (SEQ ID sense NO: 25) α-factor TATTGCCAGCATTGCTGC (SEQ ID NO: 26) 5′-AOX1 Sense GACTGGTTCCAATTGACAAGC (SEQ ID NO: 27) 3′-AOX1 Anti- GCAAATGGCATTCTGACATCC (SEQ ID sense NO: 28) RLDDL-1 Sense CAAGACTTGACGACCTAGAC (SEQ ID NO: 74) RLDD-2 Anti- GTCGTCAAGTCTTGGGATTC (SEQ ID sense NO: 75) RGDDL-1 Sense CAAGACTTGACGACCTAGAC (SEQ ID NO: 76) ARLDD-2 Anti- GTCGTCAAGTCTTGCGATTC (SEQ ID sense NO: 77)

Note: Primer α-factor was used as a sequencing primer; 5′-AOX1 & 3′-AOX1 primers were used for checking for the presence of the inserted DNA.

The polymerase chain reactions were carried out at 95° C. for 1 min, 55° C. for 1 min, then 72° C. for 1 min for 25 cycles. A mixture of primers was also used for generating multiple mutation sites. The PCR products were separated on 2% agarose gels and visualized by ethidium bromide staining. The desired PCR products were purified and then ligated into the Eco RI and Sac II sites of the yeast transfer vector pPICZα A. The recombinant plasmid was used to transform an Escherichia coli XL1-blue strain and colonies selected on agar plates containing antibiotic Zeocin. The E. coli XL1-blue colonies were picked, plasmid DNA isolated, and the sequence confirmed by sequencing the insert. Table 2 lists primer sequence ID NOs. used for synthesizing DNAs encoding Rhodostomin and various variants.

TABLE 2 Primers for synthesizing DNAs encoding Rhodostomin and various variants Antisense Sense Primer Primer Rhodostomin Variant SEQ ID NO: 3 SEQ ID NO: 4 (Rho) ⁴⁸PRGDMP⁵³ SEQ ID NO: 16 SEQ ID NO: 13 Variant ⁴⁸ARGDDP⁵³ SEQ ID NO: 24 SEQ ID NO: 13 Variant ⁴⁸ARGDDV⁵³ SEQ ID NO: 70 SEQ ID NO: 71 Variant ⁴⁸ARGDDL⁵³ SEQ ID NO: 20 SEQ ID NO: 21 Variant ⁴⁸PRGDDL⁵³ SEQ ID NO: 72 SEQ ID NO: 73 Variant ⁴⁸ARGDDM⁵³ SEQ ID NO: 22 SEQ ID NO: 23 Variant ⁴⁸PRGDDM⁵³ SEQ ID NO: 5 SEQ ID NO: 6 Variant ⁴⁸PRLDMP⁵³ SEQ ID NO: 74 SEQ ID NO: 75 Variant ⁴⁸PRLDDL⁵³ SEQ ID NO: 76 SEQ ID NO: 77 Variant ⁴⁸ARLDDL⁵³ SEQ ID NO: 9 SEQ ID NO: 10 Variant ⁴⁸PRIDMP⁵³ SEQ ID NO: 7 SEQ ID NO: 8 Variant ⁴⁸PRHDMP⁵³ SEQ ID NO: 14 SEQ ID NO: 15 Variant ⁴⁸PRGDNP⁵³ SEQ ID NO: 18 SEQ ID NO: 19 Variant ⁴⁸PRGDGP⁵³

EXAMPLE 2 Protein Expression and Purification of Rhodostomin and Variants

The protein expression of rhodostomin and its variants in Pichia was performed according to the protocols of the Pichia EasyComp™ Kit with minor modifications. Briefly, a total of 10 μg plasmids containing DNA encoding rhodostomin or its variants were purified and digested with Sac I to linearize the plasmids. Pichia strain X33 was transformed with the linearized constructs by a heat shock method, using a Pichia EasyComp™ kit from Invitrogen®. The transformant integrated at the 5′ AOX1 locus by a single crossover. PCR was used to analyze Pichia integrants to determine if the Rho gene had been integrated into the Pichia genome, and cells were lysed by Lyticase (Sigma). Colonies were selected on agar plates containing YPD (1% yeast extract, 2% peptone, 2% glucose, and 2% agar) and 100 μg/ml Zeocin. A number of clones with multiple copies of Rho gene insertions were selected to pick the clone having the highest Rho protein expression.

Recombinant Rho and its variants were produced as follows: Selected colonies were grown in the YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) containing 100 μg/ml Zeocin™ at 30° C. After 48 hours, cells were collected by centrifugation and grown in 1 liter of minimal methanol medium (containing 1.34% yeast nitrogen base with ammonium sulfate without amino acids and 4×10⁻⁵% biotin). A total of 1% methanol was added once every 24 hours to induce Rho or variant expression for 2 days. The supernatant was collected by centrifugation and dialyzed twice against 5 liter buffer A (5 mM EDTA, 8M urea and 10 mM Na-phosphate buffer, pH 7.7). The final solution was loaded into a nickel-chelating column and eluted with a gradient of 200 mM imidazole. The recombinant rhodostomin and its variants were further purified by HPLC (reverse phase C18 HPLC). The purified recombinant Rho had a purity of greater than 95% as judged by Tricine-SDS-PAGE.

Rho and its variants were subsequently subjected to electrospray mass spectrometry analyses for checking the molecular weight. The amino acid sequences of the RGD motifs of Rho and variants are shown in Table 3.

TABLE 3 SEQ ID NO. RGD Motif and Variants thereof Abbreviation 2 ⁴⁹Arg-Gly-Asp⁵¹ ⁴⁹RGD⁵¹ 29 ⁴⁸Pro-Arg-Gly-Asp-Met-Pro⁵³ ⁴⁸PRGDMP⁵³ (Wild type) 30 ⁴⁸Ala-Arg-Gly-Asp-Asp-Pro⁵³ ⁴⁸ARGDDP⁵³ 31 ⁴⁸Ala-Arg-Gly-Asp-Asp-Val⁵³ ⁴⁸ARGDDV⁵³ 32 ⁴⁸Ala-Arg-Gly-Asp-Asp-Leu⁵³ ⁴⁸ARGDDL⁵³ 33 ⁴⁸Pro-Arg-Gly-Asp-Asp-Leu⁵³ ⁴⁸PRGDDL⁵³ 34 ⁴⁸Ala-Arg-Gly-Asp-Asp-Met⁵³ ⁴⁸ARGDDM⁵³ 35 ⁴⁸Pro-Arg-Gly-Asp-Asp-Met⁵³ ⁴⁸PRGDDM⁵³ 36 ⁴⁸Pro-Arg-Leu-Asp-Met-Pro⁵³ ⁴⁸PRLDMP⁵³ (RD)(RLD) 36 ⁴⁸Pro-Arg-Leu-Asp-Met-Pro⁵³ *⁴⁸PRLDMP⁵³-5K (pegylated) 37 ⁴⁸Pro-Arg-Leu-Asp-Asp-Leu⁵³ ⁴⁸PRLDDL⁵³ 38 ⁴⁸Ala-Arg-Leu-Asp-Asp-Leu⁵³ ⁴⁸ARLDDL⁵³ 39 ⁴⁸Pro-Arg-Ile-Asp-Met-Pro⁵³ ⁴⁸PRIDMP⁵³(RID) 40 ⁴⁸Pro-Arg-His-Asp-Met-Pro⁵³ ⁴⁸PRHDMP⁵³ 41 ⁴⁸Pro-Arg-Gly-Asp-Asn-Pro⁵³ ⁴⁸PRGDNP⁵³ 42 ⁴⁸Pro-Arg-Gly-Asp-Gly-Pro⁵³ ⁴⁸PRGDGP⁵³(PGP) *⁴⁸PRLDMP⁵³-5K refers to pegylated ⁴⁸PRLDMP⁵³.

EXAMPLE 3 Effects of RD and its Derivatives on Bleeding Time

Measurement of bleeding time was performed as follows: Mice were anaesthetized with trichloroacetaldehyde (200 mg/kg), and bleeding time was measured by a method described previously, with minor modifications. Saline or proteins were injected intravenously through the tail vein of the mouse (ICR, male, with an average body weight of 23.5±1.8 g). A sharp cut of 0.5 mm from the tail tip of the mouse was made 5 min after injection. The tail was then immediately immersed in a saline-filled beaker, kept at 37° C., and the bleeding time was measured.

FIG. 1 shows the effects of RD and ARLDDL proteins on tail bleeding time in mice. Tail bleeding time was measured 5 min after intravenous administration of saline, RD, ARLDDL, rhodostomin (0.6 mg/kg for each) of HSA-RD (5 mg/kg). An intravenous injection of rhodostomin (0.5 mg/kg) exerted a pronounced effect in prolonging the clotting time in mice. However, both RD and ARLDDL (0.5 mg/kg), which exerted selectivity for αvβ3 integrin, only slightly affected the clotting time in mice as compared to rhodostomin. Results are expressed as the mean±S.E.M (n=6).

EXAMPLE 4 Platelet aggregation Assay

Venous blood (9 parts) samples from healthy donors who had not received any medication for at least two weeks were collected in 3.8% sodium citrate (1 part). Blood samples were centrifuged at 150×g for 10 min to obtain platelet-rich plasma (PRP) and allowed to stand for 5 min, and PRP was collected. The platelet-poor plasma (PPP) was prepared from the remaining blood by centrifuging at 2000×g for 25 min. The PPP platelet count was measured on a hematology analyzer and diluted to 250,000 platelets/μl. A solution of 190 μl of PRP and 10 μl of either Rho or PBS buffer were incubated for 5 min in a Hema Tracer 601 aggregometer at 37° C. Ten microliters of 200 μM adenosine diphosphate (ADP) were further added to monitor the response of platelet aggregation by light transmission.

EXAMPLE 5 Cell Adhesion Inhibition Assay

Cell adhesion inhibition assays were performed as described previously in Zhang, X. P., Kamata, T., Yokoyama, K., Puzon-McLaughlin, W., and Takada, Y. (1998) Specific interaction of the recombinant disintegrin-like domain of MDC-15 (metargidin, ADAM-15) with integrin alphavbeta3, J Biol Chem 273:7345-7350. Briefly, wells of 96-well Immulon-2 microtiter plates (Costar, Corning, USA) were coated with 100 μl of phosphate-buffered saline (PBS: 10 mM phosphate buffer, 0.15M NaCl, pH 7.4) containing substrates at a concentration of 50-500 nM, and incubated overnight at 4° C. The substrates and their coating concentrations were fibrinogen (Fg) 200 μg/ml, vitronectin (Vn) 50 μg/ml, and fibronectin (Fn) 25 μg/ml. Non-specific protein binding sites were blocked by incubating each well with 200 μl of heat-denatured 1% bovine serum albumin (BSA, Calbiochem) at room temperature (25° C.) for 1.5 hr. The heat-denatured BSA was discarded and each well was washed twice with 200 μl of PBS.

Chinese hamster ovary (CHO) cells expressing αvβ3 (CHO-αvβ3) and αIIbβ3 (CHO-αIIbβ3) integrins were maintained in 100 μl of Dulbecco's Modified Eagle's Medium (DMEM) medium. Chinese hamster ovary (CHO) cells expressing integrins αvβ3 (CHO-αvβ3) and αIIbβ3 (CHO-αIIbβ3) were kindly provided by Dr. Y. Takada (Scripps Research Institute). Human erythroleukemia K562 cells were purchased from ATCC and cultured in the RPMI-1640 medium containing 5% fetal calf serum. CHO and K562 cells growing in log phase were detached by trypsinization and used in the assay at 3×10⁵ and2.5×10⁵ cells/ml, respectively. Rho and its variants were added to the cultured cells and incubated at 37° C., 5% CO₂ for 15 minutes. Rho and its variants were used as inhibitors at the concentrations of 0.001-500 μM. The treated cells were then added into the coated plate and reacted at 37° C., 5% CO₂ for 1 hour. The incubation solution was then discarded and non-adhered cells were removed by washing twice with 200 μl PBS. Bound cells were quantified by crystal violet staining. Briefly, the well was fixed with 100 μl of 10% formalin for 10 minutes and dried. Fifty microliters of 0.05% crystal violet were then added into the well at room temperature for 20 minutes. Each well was washed with 200 μl of distilled water four times and dried. Colorization was carried out by adding 150 μl of colorizing solution (50% alcohol and 0.1% acetic acid). The resulting absorbance was read at 600 nm and the readings were correlated with the number of adhering cells. Inhibition was defined as % inhibition=100−[OD₆₀₀ (rhodostomin variant-treated sample)/OD₆₀₀ (untreated sample)]×100.

EXAMPLE 6 Inhibitory Effects of RD and its Derivatives on Integrins αvβ3, αIIbβ3, and α5β1

The IC₅₀ of RD and its derivatives on integrin binding was obtained by the cell adhesion assay as described in Example 5 above. Briefly, matrix proteins such as fibronectin, vitronectin, or fibrinogen, were coated at a fixed concentration on microtiter plates, as described in Example 5, and Rho and its variants were added to the cells expressing integrin at different concentrations ranging from 0.001-500 μM to obtain an IC₅₀. The lower the IC₅₀, the greater the specificity or potency of the variant.

The modification of the RGD motif of Rho had unique effects on Rho's biological activities: the activity in inhibiting the integrin αIIbβ3 or α5β1's binding to the matrix proteins was reduced and the selectivity to the integrin αvβ3 was increased as a result of the sequence modification. Table 4 shows the result of IC₅₀ for inhibition of cell adherence by Rho and its derivatives.

TABLE 4 Inhibition of Cell adhesion to ECM by RD and its Derivatives RGD motif variant & IC50 (nM) SEQ ID NO: αllbβ3/Fg αvβ3/Fg α5β1/Fn ⁴⁸PRGDMP⁵³ (29)    21.0 ± 11.2 (17^(a))  13.0 ± 5.7 (20)   256.8 ± 87.5 (21) ⁴⁸ARGDDP⁵³ (30)   850.9 ± 322.6 (3)  45.3 ± 17.4 (3)  5044.5 ± 1554.5 (3) ⁴⁸ARGDDV⁵³ (31)   255.2 ± 107.2 (3)  15.8 ± 5.5 (3)   213.1 ± 74.4 (3) ⁴⁸ARGDDL⁵³ (32)  1518.1 ± 740.4 (4)  41.3 ± 16.0 (5)   526.7 ± 200.3 (5) ⁴⁸PRGDDL⁵³ (33)  1224.0 ± 231.2 (4)  76.0 ± 16.0 (4)  3017.0 ± 801.5 (3) ⁴⁸ARGDDM⁵³ (34)   627.2 ± 317.6 (3)  49.0 ± 19.2 (6)   350.8 ± 81.0 (4) ⁴⁸PRGDDM⁵³ (35)  1117.8 ± 379.7 (3) 211.0 ± 91.3 (5)  4047.3 ± 1784.3 (4) ⁴⁸PRLDMP⁵³ (36)  2219.8 ± 996.9 (6)  35.0 ± 14.1 (5)  3043.3 ± 1117.6 (4) *⁴⁸PRLDMP⁵³-5K  2408.0 ± 1090.0 (2) 114.8 ± 51.1 (4) >3730 (3) ⁴⁸PRLDDL⁵³ (37) >5880 (3) 342.3 ± 110.1 (3) >5880 (4) ⁴⁸ARLDDL⁵³ (38) 59217.8 ± 966.4 (3)  36.8 ± 12.8 (3) 23171.0 ± 925.5 (3) ⁴⁸PRIDMP⁵³ (39) >5880 (2) 119.9 ± 19.7 (3) >5880 (3) ⁴⁸PRHDMP⁵³ (40)  2109.0 ± 810.0 (3) 131.6 ± 35.3 (3) >5880 (3) ⁴⁸PRGDNP⁵³ (41)   185.0 ± 61.7 (4)  24.8 ± 13.1 (5)   357.0 ± 80.6 (3) ⁴⁸PRGDGP⁵³ (42)  2591.0 ± 572.2 (3) 204.1 ± 87.0 (3)  3462.0 ± 1525.5 (4) *⁴⁸PRGDMP⁵³ (29) refers to the RGD motif of Rho and comprises an amino acid sequence set forth by SEQ ID NO: 29; ⁴⁸PRLDMP⁵³ (36) refers to the RGD motif variant of RD and comprises an amino acid sequence set forth by SEQ ID NO: 36. ^(a)The numbers represents the numbers of experiments.

The rhodostomin variants have a much lower affinity to αIIbβ3 and/or α5β1 as compared to the Rho (Table 4). As shown in Table 4, for example, the IC₅₀ of RD (i.e., PRLDMP) in inhibition of integrins αIIbβ3 and α5β1 increased more than 104 and 10-fold, respectively, as compared to that of the Rho. Moreover, the IC₅₀ of ARLDDL in inhibition of integrins αIIbβ3 and α5β1 increased more than 2000-fold compared to that of the Rho. The IC₅₀ of pegylated RD and human albumin-conjugated RD on αIIbβ3 binding increased 113.7- and 129.9-fold, respectively, as compared to that of the Rho (Table 5). Therefore, the variants' affinities to platelets were markedly reduced compared to that of Rho (Table 5).

EXAMPLE 7 Effects of RD and its Derivatives on Osteoclastogenesis

Osteoclasts are specialized monocyte/macrophage family members that differentiate from bone marrow hematopoietic precursors. Cultures of osteoclast precursors in the presence of M-CSF (20 ng/ml) and sRANKL (50 ng/ml) for 8 days induced the formation of large mature osteoclasts with multi-nuclei, which were characterized by the acquisition of mature phenotypic markers, such as TRAP. The method of osteoclastogenesis from cultured hematopoietic cells of bone marrow and the effects of RD and its derivatives on osteoclastogenesis were investigated as follows.

Bone marrow cells were prepared by removing femurs from 6˜8-week-old SD rats and flushing the bone marrow cavity with a-MEM which was supplemented with 20 mM HEPES and 10% heat-inactivated FCS, 2 mM-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml). The non-adherent cells (hematopoietic cells) were collected and used as osteoclast precursors after 24 hr. Cells were seeded at 1×10⁶ cells/well (0.5 ml) in 24-well plates in the presence of human recombinant soluble RANKL (50 ng/mI) and murine M-CSF (20 ng/ml). The culture medium was replaced every 3 days. Osteoclast formation was confirmed by an assay of tartrate-resistant acid phosphatase (TRAP) on day-8. In brief, adherent cells were fixed with 10% formaldehyde in phosphate-buffered saline for 3 min. After treatment with ethanol/acetone (50:50 v/v) for 1 min, the cell surface was air-dried and incubated for 10 min at room temperature in an acetate buffer (0.1 M sodium acetate, pH 5.0) containing 0.01% naphthol AS-MX phosphate (Sigma) and 0.03% fast red violet LB salt (Sigma) in the presence of 50 mM sodium tartrate. Osteoclast-like TRAP-positive cells in each well were scored by counting the number of TRAP-positive and multinucleated cells containing more than three nuclei.

RD derivatives markedly inhibited the differentiation of osteoclasts, which was correlated with their inhibitory activities on αvβ3 (Table 5). On the other hand, AKGDWN and PRGEMP were less effective in inhibiting integrin αvβ3 and differentiation of osteoclasts (Table 5).

TABLE 5 Inhibition of Platelet Aggregation, Cell adhesion, and Osteoclastogenesis by RD and its Derivatives IC50 (nM) Platelet aggregation αvβ3 α5β1 αIIbβ3 Osteoclastogenesis Rhodostomin 83 13 257 21 5.52 ARGDDL 455 41 527 1518 8.02 PGP 283 24 4000 500 5.76 RD 433 35 3043 2220 3.32 RID 448 120 >5850 >5850 5.82 PRGDDL 396 76 3017 1224 3.15 ARLDDL 10380 37 23171 59217 — PRPDDL 3530 188 21381 13590 AKGDWN 138 96637 >119000 69 78400 PRGEMP 4908 >17850 >5950 >59500 68200 Pegylated-RD 398 115 >5880 2408 3.88 HSA-RD 438 45 >5880 2750 6.68

EXAMPLE 8 Ovariectomy-Induced Osteoporosis

Female Sprague-Dawley rats (3 months old, 270˜290 g) or ICR mice (4 weeks old, 22˜28 g) were used for this study. Rats or mice were ovariectomized (OVX) bilaterally under trichloroacetaldehyde anesthesia and control rats were sham-operated (Sham) for comparison. The animals were all kept under controlled conditions at the room temperature (22±1° C.) and a 12-hr light-dark cycle. Animals were fed with Purina Laboratory Rodent Diet (PMI; St. Louis, Mo.) (0.95% calcium) and water ad libitum. The body weights of the rats were recorded weekly.

EXAMPLE 9 Analysis of Bone Mineral Density (BMD) and Content (BMC)

At the end of the experiment, rats or mice were sacrificed by decapitation. The tibia and femur were removed, cleaned of soft tissue, and the length and weight of the tibia and femur were measured with a precision caliper (±0.05 mm) as described by Weinreb M, Rodan G A, Thompson D D., (1991) Depression of osteoblastic activity in immobilized limbs of suckling rats, J Bone Miner Res 6:725-731. BMD and BMC of the tibia were measured with a dual-energy X-ray absorptiometer (DEX, XR-26; Norland, Fort Atkinson, Wis.). The mode adapted to the measurements of small subjects was adopted. See Chih-Hsin et al., “Enhancement of Fibronectin Fibrillogenesis and Bone Formation by Basic Fibroblast Growth Factor via Protein Kinase C-Dependent Pathway in Rat Osteoblasts,” Mol. Pharmacol: 66: 440-449, (2004). A coefficient of variation of 0.7% was calculated from daily measurements of BMD on a lumbar phantom for more than 1 year [22, 23]. The whole tibia and femur were scanned and BMD and BMC were measured by an absorptiometer.

EXAMPLE 10 Histomorphometry of Bone

Tibiae were fixed by 4% formaldehyde and then decalcified with 12% EDTA and dehydrated in an ascending series of ethanol solution and acetone, and embedded in paraffin. Serial sections (5 mm) were cut longitudinally and stained with Mayer's hematoxylin-eosin solution. Images of the growth plate and proximal tibia were photographed using an Olympus microscope. The bone volume was measured using an image analysis software (Image-pro plus 3.0) in the secondary spongiosa, which was located under the primary spongiosa and characterized by a network of larger trabeculae. To measure the number of osteoclasts, the sections were stained with tartrate-resistant acid phosphatase (TRAP).

EXAMPLE 11 Biomechanical Three-Point Bending Test

Mechanical properties of bone tissues were measured by performing three-point bending test in a material testing system (MTS-858, MTS System Inc., Minneapolis, Minn.). The span of the two support points was 20 millimeters and the deformation rate was 1 mm/min. Load/deformation curves were input to a computer and analyzed by Team 490 software (version 4.10, Nicolet Instrument Technologies Inc., Madison, Wis.). Cross-sectional parameters were measured from the photographs and used in the calculation of the cross-sectional moment of inertia. The cross-sectional moment of inertia was calculated under the assumption that the cross-sections were elliptically shaped [24]:

I=π[(ab3×(a×2t)(b×2t)3)/64

where a is the width of the cross section in the mediolateral direction, b is the width of the cross section in the anteroposterior direction, and t is the average of the cortical thickness. All of these parameters were obtained using the image software Image Pro Plus 3.0 for Windows (Media Cybernetics, Silver Spring, Md.). The maximal stress, ultimate stress, and elastic modulus (Young's modulus) were calculated using the following equations [25]:

σ=FLc/41

E=F/d

L3/48I

where σ is ultimate stress, c is the distance from the center of mass (equal to ½ b as described above), F is the applied load (N), d is the displacement (mm), and L is the span between the two support points of the bending fixture (mm). In addition, the energies in the ultimate stress were measured by computing the respective areas under the stress-strain curve.

EXAMPLE 12 Inhibition of OVX-Induced Bone Loss by RD Derivatives in Mice

To examine the effects of RD derivatives on bone loss, osteoporosis was induced in female mice by ovariectomy (OVX), as described in Example 8. OVX mice showed a decrease in BMD and BMC of total body. Treatment with RD derivatives (I.M., 1.5 mg/kg/alternate day) or alendronate (p.o., 1.5 mg/kg/alternate day) for 2 weeks inhibited the loss of BMD and BMC (Table 6). The blood concentration of the C-terminal telopeptide of collagen can reflect the osteoclastic activity. As shown in Table 6, RD derivatives or alendronate also inhibited ovariectomy-induced increase in osteoclast activity (Table 6). It appeared that some of the RD derivatives were much more effective than alendronate. In addition, treatment with RD (I.M., 1.5 mg/kg) once per week for 2 weeks also inhibited the loss of BMD and BMC (Table 6). These data indicate that RD and its derivatives may inhibit osteoporosis at longer dosing intervals.

TABLE 6 Inhibition of OVX-induced bone loss by RD and its derivatives in mice C-terminal telopeptide of COL(1)α1 BMD (g/cm²) BMC (g) chain (ng/ml) (Alternate day) Sham 0.095 ± 0.001 0.649 ± 0.011 395 ± 11.9 OVX 0.075 ± 0.002* 0.517 ± 0.007* 686 ± 12.1* OVX + RD 0.091 ± 0.001§ 0.627 ± 0.008§ 391 ± 8.3§ OVX + PGP 0.092 ± 0.001§ 0.621 ± 0.006§ 372 ± 24.2§ OVX + ARGDDL 0.093 ± 0.001§ 0.624 ± 0.007§ 389 ± 16.4§ OVX + RID 0.091 ± 0.001§ 0.622 ± 0.004§ 391 ± 12§ OVX + Rhodostomin 0.090 ± 0.001§ 0.619 ± 0.006§ 397 ± 13.6§ OVX + PRLDDL 0.091 ± 0.002§ 0.563 ± 0.056§ 410 ± 25.1§ OVX + Alendronate 0.086 ± 0.002§ 0.607 ± 0.051§ 504 ± 19.6§ OVX + pegylated-RD 0.092 ± 0.002§ 0.629 ± 0.008§ 398 ± 9.2§ (Once/week) OVX + RD 0.085 ± 0.002§ 0.582 ± 0.009§ 533 ± 27.4§ OVX + pegylated-RD 0.087 ± 0.005§ 0.595 ± 0.009§ 538 ± 19.8§ Values are means ± SE. *Compared with Sham-group, p < 0.05 §Compared with OVX-group, p < 0.05

EXAMPLE 13 Inhibition of Ovariectomy-Induced Bone Loss by PGP and RD Derivatives in Rats

PGP (a RD derivative) was chosen to examine in more detail the protection from ovariectomy (OVX)-induced bone loss in rats. Adult female rats (3 month-old) were ovariectomized, as in Example 8, and bone volumes were measured 6 weeks after the ovariectomy, as in Examples 9-11. It was shown that PGP protein inhibited both ovariectomy-induced bone volume decrease and osteoclast number increase.

As shown in FIG. 2A, compared to the sham-operated rats (Sham), ovariectomy (OVX) caused a significant loss of the trabecular bone. However, treatment with PGP (IV, 0.3 mg/kg/day or IM, 1.5 mg/kg/alternate day) significantly inhibited the ovariectomy-induced loss of the trabecular bone in secondary spongiosa.

In FIG. 2B, tartrate-resistant acid phosphatase (TRAP) staining showed that osteoclasts were predominantly localized around the trabecular bone, and that PGP inhibited the OVX-induced increase in osteoclast formation.

OVX rats also showed an increase in body weight at the end of the experiment. Treatment with PGP (I.V., 0.3 mg/kg/day or I.M., 1.5 mg/kg/alternate day) significantly inhibited the OVX-induced increase in body weight.

Rats also showed a decrease in wet weight, BMD and BMC in both femur and tibia 6 weeks after the ovariectomy (Table 7). Treatment with PGP (either I.V. or I.M.) inhibited the reduction in wet weight, BMD and BMC of both tibia and femur in OVX rats. Histomorphometry demonstrated that ovariectomy caused the loss of trabecular bone in secondary spongiosa (FIG. 2). Treatment with PGP markedly reversed the loss of bone volume (FIG. 2 and Table 7). In addition, TRAP staining demonstrated that osteoclasts were predominantly localized around the trabecular bone and OVX increased the osteoclast number. A long-term administration of PGP antagonized the OVX-induced osteoclastic formation (FIG. 2 and Table 7). The serum level of the C-terminal telopeptide of collagen reflects osteoclast activity. It was found that the value was markedly increased in the OVX-group compared with that of the Sham-group, whereas treatment with PGP effectively antagonized the OVX-induced increase in osteoclast activity (Table 7).

TABLE 7 Inhibition of Ovariectomy-induced Bone Loss by PGP Protein in Rats OVX + PGP (IV) OVX + PGP (IM) Sham (n = 22) OVX (n = 22) (n = 13) (n = 12) Bone length, mm Tibia 4.04 ± 0.01 4.04 ± 0.01  4.05 ± 0.01  4.04 ± 0.01  Femur 3.62 ± 0.01 3.62 ± 0.02  3.60 ± 0.02  3.64 ± 0.02  Wet weight, mg Tibia 755.5 ± 6.3  647.5 ± 8.5*  735.8 ± 8.5§  740.6 ± 8.7§  Femur 946.0 ± 10.9  850 ± 9.9*  904.9 ± 10.2§  904.0 ± 13.5§  BMD, g/cm² Tibia 0.108 ± 0.002 0.099 ± 0.015* 0.109 ± 0.002§ 0.111 ± 0.002§ Femur 0.131 ± 0.012 0.126 ± 0.003* 0.130 ± 0.002§ 0.136 ± 0.002§ BMC, g Tibia 0.301 ± 0.011 0.269 ± 0.011* 0.286 ± 0.010§ 0.316 ± 0.009§ Femur 0.420 ± 0.005 0.369 ± 0.011* 0.422 ± 0.013§ 0.437 ± 0.009§ Bone volume, % 18.1 ± 1.5  9.4 ± 1.5* 14.3 ± 1.5§  16.8 ± 1.8§  N.Oc/BS (No/mm) 1.77 ± 0.09 2.06 ± 0.17* 1.83 ± 0.10§ 1.81 ± 0.09§ C-terminal 230 ± 5.1   459 ± 18.4*  259 ± 22.8§  221 ± 15.1§ telopeptide of collagen (n = 5 for each) ALP, alkaline phosphatase; N.Oc/BS, osteoclast number/mm bone surface. Values are means ± SE. *Compared with Sham-group, p < 0.05 §Compared with OVX-group OVX, p < 0.05

A three-point bending test was performed in the femur to examine the mechanical activity of the bone. Compared with the Sham-operated group, the maximal load, ultimate load, Young's modulus and ultimate stress decreased in OVX rats. Treatment with PGP exhibited a protection against the OVX-induced decrease in bone strength (Table 8). These results suggest that PGP-like protein drugs may markedly inhibit the bone loss caused by ovariectomy.

TABLE 8 Increase in biomechanical properties by PGP protein in OVX rats Sham OVX + PGP OVX + PGP (n = 22) OVX (n = 22) (IV) (n = 13) (IM) (n = 12) Maximal load, N 132.7 ± 4.8 112.1 ± 4.3* 123.9 ± 4.9§ 129.8 ± 7.9§ Ultimate loading, N  85.5 ± 3.7  70.4 ± 3.1*  81.9 ± 2.2§  83.2 ± 2.5§ Young's modulus, GPa 203.7 ± 3.6 185.5 ± 3.3* 194.0 ± 3.0§ 199.9 ± 6.2§ Energy to ultimate  5.9 ± 0.4  3.5 ± 0.3*  4.9 ± 0.8§  5.1 ± 0.9§ stress, mJ/mm³ Values are means ± SE. *Compared with Sham-group, p < 0.05 §Compared with OVX-group OVX, p < 0.05

EXAMPLE 14 Post-Treatment of RD Protein Inhibited OVX-Induced Osteoclast Activation

To examine the dynamic therapeutic effect of RD derivatives on osteoclast activity in vivo, osteoporosis was induced in female rats by ovariectomy and the C-terminal telopeptides of type-I collagen were measured from blood at different intervals. Compared with sham-operated rats (Sham), ovariectomy (OVX) caused a significant increase in the osteoclast activity. Measurement of the serum level of the C-terminal telopeptide of COL(1)α1 chain showed an increase in the osteoclast activity in OVX rats. The C-terminal telopeptide of type-I collagen increased from a basal level of 361±25.6 (n=15) to 708±50.7 ng/ml (n=15) 28 days after the ovariectomy.

RD or alendronate was then post-treated one month after the ovariectomy. The post-treatment with RD (I.M., 1.5 mg/kg/alternate day) or alendronate (p.o., 1.5 mg/kg/alternate day) reversed the ovariectomy-induced increase in osteoclast activity, and RD was much more effective than alendronate (FIG. 3). Osteoclast activity recovered after the withdrawal of the RD or alendronate treatment. The recovery of osteoclast activity was much faster in the alendronate-treated group than the RD-treated group. However, re-application of RD effectively inhibited the ovariectomy-induced increase in osteoclast activity. The value of the osteoclast in response to the RD treatment was much lower than that in response to the alendronate treatment (FIG. 3). These results indicate that RD-related proteins have therapeutic effects on the bone loss caused by ovariectomy-induced osteoclastic activation.

After the second drug treatment period, rats were sacrificed, and BMD and BMC of the tibia and femur were measured. As shown in Table 9, post-treatment of RD effectively reversed the bone loss effect of ovariectomy. With regard to the preservation of the bone volume, RD was more effective than alendronate.

TABLE 9 Post-treatment of RD inhibits OVX-induced bone loss in rats Sham OVX RD Alendronate Bone length, mm Tibia 4.04 ± 0.03 4.05 ± 0.03  4.01 ± 0.03  4.02 ± 0.05 Femur 3.69 ± 0.03 3.68 ± 0.02  3.71 ± 0.06  3.68 ± 0.05 Wet weight, mg Tibia 766.6 ± 19.9   661 ± 18.2*  749 ± 20.1§ 712.1 ± 10.1§ Femur 1002.6 ± 17.4   861 ± 10.7* 985.3 ± 9.8§  912.4 ± 10.9§ BMD, g/cm² Tibia 0.114 ± 0.003 0.101 ± 0.002* 0.113 ± 0.009§  0.108 ± 0.002§ Femur 0.137 ± 0.002 0.121 ± 0.002* 0.135 ± 0.004§  0.131 ± 0.003§ BMC, g Tibia 0.315 ± 0.013 0.272 ± 0.006* 0.299 ± 0.011§  0.289 ± 0.009§ Femur 0.442 ± 0.012 0.391 ± 0.008* 0.441 ± 0.018§  0.401 ± 0.017§ Bone volume, % 18.1 ± 1.2  7.9 ± 2.1* 16.3 ± 2.7§  12.1 ± 1.3§ BMD is the abbreviation of bone mineral density; BMC is the abbreviation of bone mineral content. Values are means ± SD. *Compared with Sham-group, P < 0.05 §Compared with OVX-group OVX, P < 0.05 RD: I.M. injection (1.5/mg/kg/alternate day) Alendronate: p.o. (1.5 mg/kg/alternate day) n = 5 for each group

EXAMPLE 15 Inhibition of Chondrocyte Damage by RD in Osteoarthritis Animals

An osteoarthritis animal model was obtained by surgery only on the right knees of male Sprague-Dawley rats. Surgery involved anterior cruciate ligation transaction and partial medial meniscectomy. After the surgery, RD protein was administered via intramuscular route (1.5 mg/kg/alternate day) or local joint injection (once/week) until the last day when the rats were sacrificed 6 weeks after the surgery. Each joint was embedded in paraffin wax, sectioned and stained with 0.1% safranin-O and haematoxylin. As shown in FIG. 4B, the chondrocyte layer in the right knee joint was damaged by arthritis and RD in either intramuscular injection (FIG. 4D) or local application (FIG. 4C) inhibited the chondrocyte layer destruction.

EXAMPLE 16 Inhibition of Blood Cytokine Elevation by RD in Osteoarthritis Rat

Osteoarthritic rats were generated by surgery as described above. Six weeks after surgery, serum was obtained to measure the blood level of cytokines. As shown in FIGS. 5A-5C, the serum levels of cytokines, such as c-reactive protein, IL-1β and IL-6, increased in osteoarthritic rats, and RD administration markedly inhibited the osteoarthritis-induced elevation of cytokines.

EXAMPLE 17 Inhibition of Blood Cytokine Elevation by RD in Osteoarthritis Mice

Osteoarthritic mice were generated by surgery as described above. Six weeks after the surgery, the serum was obtained to measure the level of cytokines. As shown in FIG. 6A-6C, the serum levels of cytokines, such as c-reactive protein, IL-1□ and IL-6, increased in osteoarthritic mice, and RD administration markedly inhibited the osteoarthritis-induced elevation of cytokines.

EXAMPLE 18 Intra-Tibia Injection of Prostate or Breast Cancer Cells in Mice

Severe Combined Immune Deficiency (SCID) male mice or BALB/c-nu/nu male mice weighing 20-22 g (6 weeks old) bred in an animal isolator (IVC racks) under a specific pathogen-free (SPF) condition at 22±2° C. were used. Human prostate adenocarcinoma PC-3 cells or human breast cancer MDA-MB-231 cells (1×10⁶ cells in 15 ml sterile PBS) were injected into the bone marrow of both the left and right tibia of the SCID mice or nude mice on day-1, respectively. On day-11, the animals were randomly assigned into three groups and the administration of test substances was initiated. RD (1.5 mg/kg) was administered by intramuscular (IM) injection and alendronate (1.5 mg/kg) was given by subcutaneous (SC) injection, once daily for a total of 15 doses (5 days on, 2 days off for 3 weeks). The body weight and tumor growth condition were observed and recorded every week during the experimental period. After one month, the mice were sacrificed and the weight of the hind legs was measured. The weight of the control leg was subtracted to reflect the relative tumor weight. In addition, blood samples were collected at the end of the experiment for the blood count of red blood cells (RBC), white blood cell (WBC) and platelet, and for the measurement of the C-terminal telopeptides of type I collagen (CTX) and serum calcium concentration.

In order to determine bone osteolysis, radiographs were taken by a soft X-ray generating unit (Young-kid Enterprise Co., Ltd., Taipei, Taiwan). Animals were deeply anesthetized with trichloroacetaldehyde monohydrate, laid down in a prone position on a Kodak Scientific Imaging film (13×18 cm), and X-ray exposure was performed at 45 kV for 5 seconds. The degree of osteolysis was measured using image analysis software (Image-pro plus 3.0).

EXAMPLE 19 Inhibition of Tumor Growth in Bone and Hypercalcemia

Tumor cell growth in bone is related to the bone resorption activity. The effect of RD protein on the tumor growth of prostate cancer cells in bone was thus examined. PC-3 cells (1×10⁶) were locally injected into the bone marrow cavities of both tibia in SCID mice. RD or alendronate was administered 10 days after the implantation of tumor cells. RD at 1.5 mg/kg was given by the intramuscular (IM) route while alendronate was given at 1.5 mg/kg by the subcutaneous (SC) injection, once daily for a total of 15 doses (5 days on, 2 days off for 3 weeks). The swelling of hind legs was calculated to reflect the tumor growth on day 33. The body weight was measured throughout the experimental period. The results indicated that RD at 1.5 mg/kg caused a significant inhibition in tumor cell-induced swelling in hind legs on day-33 (43.8±4.1%, n=21-22) (FIG. 7). However, the subcutaneous administration of alendronate at 1.5 mg/kg did not inhibit the tumor growth in legs (FIG. 7).

FIG. 8 shows the effect of RD on the decrease in body weight in response to the tumor growth in SCID mice. Untreated control mice showed a decrease in body weight at the end of the experiment. Treatment with RD prevented the loss of body weight caused by the tumor growth. Alendronate was used as a positive control.

FIG. 9 shows the inhibition of tumor growth and osteolytic bone lesion by RD in SCID mice. In order to determine bone osteolysis, radiographs were taken by a soft X-ray generating unit. In FIG. 9A, photographs were taken 33 days after the intratibial injection of PC-3 cells. A visible spherical tumor grew up from the proximal tibia. Treatment with RD (I.M., 1.5 mg/kg/alternate day) inhibited tumor growth. In FIG. 9B, radiographs taken on day-33 revealed that osteolytic lesions appeared in the cancer cell-injected tibia and treatment of RD inhibited osteolysis. FIG. 9C shows the quantitation of the data. FIG. 9D shows that RD inhibited a tumor-induced increase in C-terminal telopeptides of type-collagen (a marker for the osteoclast activity) using an ELISA method. FIG. 9E demonstrates that RD and alendronate (1.5 mg/kg/alternate day) also inhibited a tumor-induced increase in the serum calcium concentration (i.e., hypercalcemia). *: p<0.05, as compared with control. #: p<0.05, as compared with the PC-3-injected group.

Breast cancer has a strong predilection for metastasizing to bone. FIG. 10 shows inhibition of tumor growth by RD in nude mice. Human breast cancer cells MDA-MB-231 (1×10⁶) were locally injected into the bone marrow cavities of both tibia in nude mice. RD (I.M., 1 mg/kg/day) was administered 10 days after the implantation of tumor cells for a total of 14 days. Treatment with RD (IM, 1.5 mg/kg/day) for 2 weeks inhibited MDA-MB-231-induced increase in tumor growth in bone (FIG. 10A). Furthermore, RD also prevented the tumor-induced increase in osteoclast activity (FIG. 10B) and hypercalcemia (FIG. 10C), but did not affect the blood counts of RBC, WBC and platelet (FIGS. 10D-10F). *: p<0.05, as compared with the control. #: p<0.05, as compared with the MDA-MB-231-injected group.

EXAMPLE 20 Pegylation of RD Protein

A pegylated product or a conjugation with albumin can prolong the duration and decrease the antigenicity of a protein drug. In order to minimize the antigenicity and prolong the duration of RD protein, pegylated RD protein was prepared as follows: RD protein (4 mg) in 20 mM NaCNBH₃ at pH 5 was reacted with 5 mM PEGk5-propionaldehyde (O-Methyl-O′-[2-(6-oxocaproylamino)ethyl]polyethylene glycol 5,000)(Sigma) at 4° C. for 12 h. The pegylated RD protein was purified by reverse phase C18 HPLC. The final yield of pegylated RD protein after purification was greater than 60%.

As shown in Table 6, pegylated-RD inhibited the differentiation of osteoclasts. In addition, treatment with pegylated-RD (I.M., 1.5 mg/kg, once/week) for 2 weeks inhibited the loss of BMD and BMC (Table 6). These data indicate that pegylated-RD did not lose its activity in vivo.

EXAMPLE 21 MATRIGEL™ Plug Antiangiogenesis Assays

It has been reported that integrin αvβ3 is related to angiogenesis. Whether RD protein can inhibit angiogenesis was investigated using MATRIGEL™ plug angiogenesis assays as described previously with minor modifications. See, Passaniti A, Taylor R M, Pili R, Guo Y, Long P V, Haney J A, Pauly R R, Grant D S, Martin G R (1992) A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor, Lab Invest 67: 519-528.

Briefly, an aliquot (500 μl) of MATRIGEL™ (Becton Dickinson Lab.) containing 200 ng/ml VEGF was injected subcutaneously into the dorsal region of 6-8 week-old C57BL/6 mice. The MATRIGEL™ formed a plug rapidly. RD was administered intramuscularly (3 mg/kg) daily (RD/1d) or the other day (RD/2d or HSA-RD) before sacrifice. After 8 days, plugs were taken and photographed (FIG. 11A). Neovessels were quantified by measuring the hemoglobin of the plugs as an indication of blood vessel formation with the Drabkin method and Drabkin reagent kit 525 (Sigma) (FIG. 11B). As shown in FIGS. 11A and 11B, RD protein was effective in inhibiting angiogenesis using MATRIGEL™ plug assays. *: P<0.05 versus control group.

EXAMPLE 22 Radioligand-Binding Assay: Specificity of RD-Related Proteins

To determine whether RD and its derivative PGP bind to other receptors besides αvβ3 integrin, RD and its derivative PGP were used to analyze the target specificity toward receptors whose ligands are proteins (assayed by MDS Pharma services, Taipei, Taiwan).

As shown in Table 10, RD and PGP did not affect the binding activity of calcitonin, endothelin ETA, endothelin ETB, insulin, leptin, sodium channel, transforming growth factor-β (TGF-β), tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) to their respective receptors. This indicates that RD-related proteins exert selective binding activity toward target protein αvβ3 in viva

TABLE 10 The binding assay of RD and PGP protein Inhibition Target RD PGP Calcitonin 4% −6% Endothelin ET_(A) 26% −3% Endothelin ET_(B) 10% −7% Insulin −6% −8% Leptin 1% 2% Sodium Channel (Site 2) 2% −14% TGF-β 14% −10% TNF 4% 20% VEGF −14% 19% TGF-β: transforming growth factor-β TNF: tumor necrosis factor VEGF: vascular endothelial growth factor

EXAMPLE 23 Inhibition of Angiogenesis by RD in a Mouse Model of Retinopathy of Prematurity

An animal model for retinopathy of prematurity in mice was generated by using hypoxic-induced angiogenesis as described in Wilkinson-Berka et al. [28]. Briefly, seven-day-old pups and their mother were housed in sealed chambers containing 75% O₂ and air. Mice remained in the chamber for five days (hyperoxic period, P7 to P12) and were then housed in room air for a further seven days (hypoxic-induced angiogenic period, postnatal 12 days to postnatal 19 days, or P12 to P19). RD (2 μg) was administered via an intravitreous route on day-12 and the mice were sacrificed on day-19.

Three sections from one of the eyes of each animal were made, deparaffinized, and stained with hematxylin and eosin. Blood vessel profiles (BVPs) were counted in the inner retina, and included vessels adherent to the inner limiting membrane. Counting was performed on a photomicroscope (Leica) at a magnification of 100×.

As shown in FIG. 12A, RD protein inhibited angiogenesis in a mouse model of retinopathy of prematurity (ROP). FIG. 12B shows reduced BVPs in a mouse model of retinopathy of prematurity (ROP) treated with RD protein. Angiogenesis was quantitated by counting blood vessel profiles (BVPs) in the inner retina and extending into the vitreous cavity from three hematoxylin-and-eosin-stained sections. The ROP group treated with RD (2 μg) (i.e., ROP RD) reduced about 46% of angiogenesis compared to the ROP group treated with vehicle (ROP). (n=7 for each ROP group; n=2 for sham group.) Data are presented as Mean±SE. #: p<0.01, as compared to the sham group. **: p<0.001, as compared to the ROP group.

EXAMPLE 24 Inhibition of Ovariectomy-Induced Osteoporosis by Albumin-Conjugated RD

The effect of albumin-conjugated RD on osteoporosis was examined in ovariectomized female mice. Human serum albumin-conjugated RD (i.e., RD-albumin) was administered as indicated by arrows shown in FIGS. 13A and 13B. The data for RD was incorporated into FIGS. 13A-13D for comparison. Serum levels of the C-terminal peptide of type I collagen and alkaline phosphatase (ALP) were measured as indicators of osteoclast and osteoblast activity, respectively. The BMD and BMC were also measured every 2 weeks, as shown in FIGS. 13C and 13D. Treatment of RD-albumin (15 mg/kg/week) markedly reduced osteoclast, but increased ALP, activities in a reversible manner.

EXAMPLE 25 Inhibition of Rheumatoid Arthritis by RD

Rheumatoid arthritis is a chronic systemic inflammatory disorder of unknown etiology characterized by invasive synovial hyperplasia that leads to progressive joint destruction. Osteoclasts, derived from the monocyte/macrophage lineage, play a crucial role in subchondral bone destructions in rheumatoid arthritis. Radiographic studies have shown that in rheumatoid arthritis, osteopenia of the subchondral bone and bone erosion begin at an early stage of the disease, and are gradually exacerbated. See, van der Heijde D M, van Leeuwen M A, van Riel P L, van de Putte L B, Radiographic progression on radiographs of hands and feet during the first 3 years of rheumatoid arthritis measured according to sharp's method (van der Heijde modification), J Rheumatol 1995; 22:1792-1796. Bone-resorbing osteoclasts are observed at the erosive synovium/bone interface. See, Sakae Tanaka (2007), Signaling axis in osteoclast biology and therapeutic targeting in the RANKL/RANK/OPG System, Am. J. Nephrol, 27:466-478. A recent review discusses the role of osteoclasts in rheumatoid arthritis. Sato K, Takayanagi H., (2006) Osteoclats, rheumatoid arthritis and osteoimmunology, Curr Opin Rheumatol 18: 419-426. Therefore, RD-related proteins, which markedly inhibit osteoclast functions, may be useful for treatment of rheumatoid arthritis.

Lewis rats are given intradermal/subcutaneous (SC) injections of bovine type II collagen (2 mg/ml in Freund's incomplete adjuvant). As rats develop the rheumatoid disnotease, they are randomly divided into several study groups. Treatment is initiated on the first day when the clinical signs of arthritis are clearly visible, as evidenced, for example, by ankle joint swelling. After measuring the paw volume, rats are sacrificed, and ankle and knee joints are collected for the examination of histopathological change.

FIGS. 14A-D show amino acid sequences of Rho and its variants, SEQ ID NOs: 1, and 57-69, respectively. FIGS. 15A-C show nucleotide sequences of rhodostomin variants, SEQ ID NOs: 43-56. FIGS. 16A-H show amino acid and nucleotide sequences of disintegrin variants, SEQ ID NOs: 78-135.

EXAMPLE 26 Pegylation of ARLDDL Protein

A pegylated product or a conjugation with albumin can prolong the duration and decrease the antigenicity of a protein drug. In order to minimize the antigenicity and prolong the duration of ARLDDL protein, pegylated ARLDDL (PEG-ARLDDL) protein was prepared substantially as pegylated-RD protein was prepared as described in Example 20.

Briefly, ARLDDL protein (4 mg) in 20 mM NaCNBH₃ at pH 5 was reacted with 5 mM PEGk5-propionaldehyde (O-Methyl-O′-[2-(6-oxocaproylamino)ethyl]polyethylene glycol 5,000) (Sigma) at 4° C. for 12 h. PEG-ARLDDL protein was purified by reverse phase C¹⁸ HPLC. The final yield of PEG-ARLDDL protein after purification was greater than 60%.

EXAMPLE 27 MATRIGEL™ Plug Anti-Angiogenesis Assays

To investigate whether PEG-ARLDDL can inhibit angiogenesis, MATRIGEL™ plug angiogenesis assays were used as described in parent US Patent Application Publication No. 2008-0188413 A1. Briefly, 0.3 ml of MATRIGEL™ (Becton Dickinson Lab.) containing 150 ng recombinant human VEGF and 30 IU heparin was injected subcutaneously into the dorsal region of 6-8 week-old C57BL/6 mice. The MATRIGEL™ formed a plug rapidly. On day 2, PEG-ARLDDL at 0.5 mg/kg or at 5 mg/kg was intravenously administered via tail. After seven days, the skin of mouse was pulled back and the Matrigel™ plug was excised from it. FIG. 17A depicts photographs of the plugs.

Neovessels were quantified by measuring the hemoglobin of the plugs as an indication of blood vessel formation with the Drabkin method and Drabkin reagent kit 525 (Sigma) (FIG. 17B).

FIGS. 17A (photographs) and 17B (a bar chart) show that PEG-ARLDDL was effective in inhibiting angiogenesis using MATRIGEL™ plug assays because it significantly reduced the hemoglobin content of the Matrigel™ plug as compared with control group. *: P<0.05 versus control

EXAMPLE 28 Dose-Dependent Inhibition of PC-3 Tumor Growth by PEG-ARLDDL

The human PC-3 (prostate cancer) cells were implanted in Non-Obese Diabetic Severe Combined Immune Deficiency (NOD-SCID) mice as follows. 4 to 5 week-old male NOD-SCID mice were purchased from Laboratory Animal Center of the National Taiwan University. All mice were kept under standard temperature, humidity, and timed lighting conditions and were provided mouse chow and water. All animal experiments were conducted in accordance with the Guidelines for Animal Research of Agriculture Council, ROC and approved by the Ethical Committee for Animal Research of the National Taiwan University.

PC-3 tumor cells at 10⁷ were injected subcutaneously in the flank of the mice in serum-free medium.

One week after PC-3 cells injection, mice were injected intravenously with saline (control, n=4); PEG-ARLDDL (2 mg/kg/week, n=2) and PEG-ARLDDL (10 mg/kg/week, n=4). Tumor volume was measured weekly. After the mice were sacrificed on day 39, tumors were isolated and weighted.

Tumor Volume was calculated using the following equation:

0.52×(width(cm)²×length).

Tumor weight was estimated based on the assumption that 1 mg is equivalent to 1 mm³ of tumor volume.

FIG. 18 depicts photographs of tumors excised from the control mice (left photograph); mice treated with 2 mg/kg/week PEG-ARLDDL (middle photograph); and mice treated with 10 mg/kg/week PEG-ARLDDL (right photograph).

FIG. 19 is a bar chart that shows that PEG-ARLDDL significantly inhibited tumor growth in the mice treated with PEG-ARLDDL in a dose-dependent manner as measured by tumor weight.

In another experiment, everything was done in the same manner as described above, except that one week after PC-3 cells injection, mice were injected intravenously with saline (control, n=4); PEG-ARLDDL (10 mg/kg/week, n=2) (once every two weeks) and PEG-ARLDDL (10 mg/kg/week, n=4) (once a week). Tumor volume was measured weekly.

FIG. 20 is a graph that shows that PEG-ARLDDL significantly inhibited growth of tumors in the mice treated with this protein as measured by tumor volume. 10 mg/kg/week PEG-ARLDDL administered once a week had a stronger inhibitory effect than 10 mg/kg/week PEG-ARLDDL administered once every two weeks.

FIG. 21 depicts photographs of tumors excised from the control mice (left photograph); mice treated with 10 mg/kg/week PEG-ARLDDL once every two weeks (middle photograph); and mice treated with 10 mg/kg/week PEG-ARLDDL once a week (right photograph). The results confirm that tumor volume was the smallest in the mice treated with 10 mg/kg/week PEG-ARLDDL once a week.

FIG. 22 is a bar chart that shows that PEG-ARLDDL significantly inhibited growth of tumors in the mice treated with this protein as measured by tumor weight. After mice were sacrificed on day 39, tumors were isolated and weighted. 10 mg/kg/week PEG-ARLDDL administered once a week had a stronger inhibitory effect than 10 mg/kg/week PEG-ARLDDL administered once every two weeks.

EXAMPLE 29 Enhancement of the Cytotoxic Activity of Daunomycin by PEG-ARLDDL

Human PC-3 (prostate cancer) cells were implanted in NOD-SCID mice as follows.

PC-3 tumor cells at 10⁷ were injected subcutaneously in the flank of the mice in serum-free medium.

One week after PC-3 cells injection, mice were injected intravenously with saline (control, n=4); daunomycin (0.05 mg/kg/week, once a week; n=3); PEG-ARLDDL alone (2 mg/kg/week, n=2) and a combination of daunomycin (0.05 mg/kg/week) and PEG-ARLDDL (2 mg/kg/week, n=2). Tumor volume was measured weekly. After the mice were sacrificed on Day 39, tumors were isolated and weighted.

FIG. 23 is graph that shows that daunomycin significantly enhanced the cytotoxic activity of PEG-ARLDDL. The tumors were smallest in the mice treated with the combinatorial treatment.

FIG. 24 depicts photographs of tumors excised from the control mice (left photograph); mice treated with daunomycin alone (second from the left photograph); mice treated with PEG-ARLDDL alone (second from the right photograph); and mice treated with a combination of daunomycin and PEG-ARLDDL (right photograph). The photographs demonstrate that the tumor was the smallest in mice treated with a combination of daunomycin and PEG-ARLDDL.

FIG. 25 a bar chart that further shows that a combination of daunomycin and PEG-ARLDDL significantly inhibited growth of tumors in the mice, as compared with mice treated with only daunomycin or only PEG-ARLDDL, as measured by tumor weight. There is a synergistic effect from combining the two treatments.

EXAMPLE 30 Inhibition of Osteopontin-Induced Glioma Invasion in Hypoxia by PEG-ARLDDL

The invasion assay was performed using Transwell (Costar, N.Y., USA; pore size 8 μm) in 24-well dishes. Filters were precoated with 30 μl MATRIGEL™ containing 100 μg/ml hyaluronic acid.

Approximately 5×10⁴ U87 human glioma cells in MEM containing 0.5% FBS were placed in the upper chamber of the Transwell and 600 μl of the same medium containing 10% FBS was placed in the lower chamber.

PEG-ARLDDL at 10 nM, at 100 nM and at 1 μM was added to the cells in the upper chamber. The plates were incubated for 15 hours at 37 degrees C. in hypoxic condition (94% N₂, 5% CO₂ and 1% O₂). Non-migratory cells on the upper surface of the filters were removed by wiping with a cotton swab. Migrated cells that penetrated through pores and migrated to the underside of the filters were stained with 0.05% crystal violet solution containing 20% methanol. The cell number per well was counted under microscope at 100× magnification.

FIG. 26 depicts photographs of the cells that show that osteopontin-induced glioma invasion in hypoxic conditions was markedly inhibited by PEG-ARLDDL. It is clear from the picture in the bottom right hand corner that there are significantly fewer glioma cells after treatment with PEG-ARLDDL at 100 nM than there are glioma cells after treatment with PEG-ARLDDL at 10 nM. The number of glioma cells is the highest when no PEG-ARLDDL was added.

FIG. 27 is a bar chart that further confirms that PEG-ARLDDL significantly inhibits glioma invasion in hypoxic condition. When additional 3 ng/ml of OPN were added, the cells which were not treated with PEG-ARLDDL had almost 2-fold increase in invasion glioma cells compared to control. The cells treated with 10 nM PEG-ARLDDL had about 1.2-fold increase in invasion glioma cells compared to control; and the cells treated with 100 nM PEG-ARLDDL had about 0.75-fold increase in invasion glioma cells compared to control. The expression of OPN can be increased in hypoxic condition (94% N₂, 5% CO₂ and 1% O₂). When no additional OPN was added, the cells treated with 100 nM PEG-ARLDDL had virtually no increase in invasion glioma cells compared to control. The cells treated with 1 μM PEG-ARLDDL combined with or without 3 ng/ml of OPN had over 2-fold decrease in invasion glioma cells compared to control.

EXAMPLE 31 Inhibition of U87 Human Glioma Tumor Growth by PEG-ARLDDL

Approximately 5×10⁴ U87 human glioma cells in MEM containing 0.5% FBS were injected subcutaneously in the flank of 4 to 5 week-old male NOD-SCID mice. One week after U87 cells injection, mice were injected intravenously with saline (control; n=1) or PEG-ARLDDL (10 mg/kg, twice per week; n=2). Tumor volume was measured weekly.

FIG. 28 contains pictures of a control mouse and its excised tumor (pictures on the left) and pictures of the mouse injected with PEG-ARLDDL and its excised tumor (pictures on the right). The pictures clearly show that PEG-ARLDDL significantly inhibited growth of U87 human glioma tumors in the mice treated with this protein. FIG. 29 is a graph that shows that PEG-ARLDDL significantly inhibited growth of U87 human glioma tumors in the mice treated with this protein.

The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. 

1. A method for treatment and/or prevention of an αvβ3 integrin-associated disease comprising: administering to a mammal in need thereof a therapeutically effective amount of an isolated polypeptide, or a pharmaceutically acceptable salt thereof, wherein the isolated polypeptide is a variant of a disintegrin which comprises a variant of SEQ ID NO: 29 that contains an amino acid substitution at position 3 and an optional substitution at one or more positions 1, 5 and 6 of SEQ ID NO:
 29. 2. The method of claim 1, wherein the disintegrin is a variant of a disintegrin chosen from rhodostomin, albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, flavoridin, flavostatin, halysin, halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, and viridin.
 3. The method of claim 2, wherein the disintegrin variant comprises a rhodostomin variant.
 4. The method of claim 1, wherein said isolated polypeptide comprises a variant of an amino acid sequence chosen from SEQ ID NOs: 36-40.
 5. The method of claim 1, wherein said isolated polypeptide comprises an amino acid sequence chosen from SEQ ID NOs: 63-67.
 6. The method of claim 1, wherein said isolated polypeptide comprises SEQ ID NO:
 38. 7. The method of claim 1, wherein said isolated polypeptide comprises SEQ ID NO:
 65. 8. The method of claim 1, wherein said isolated polypeptide is encoded by a polynucleotide comprising SEQ ID NO:
 52. 9. The method of claim 1, wherein said polypeptide is pegylated or conjugated with albumin.
 10. The method of claim 1, wherein the αvβ3 integrin-associated disease is chosen from osteoporosis, cancer, bone tumor or cancer growth and symptoms related thereto, tumor cell growth in bone, tumor metastasis in bone, malignancy-induced hypercalcemia, Paget's disease, rheumatic arthritis, osteoarthritis, and an ovariectomy-induced physiological change.
 11. The method of claim 10, wherein said symptoms comprise a pathological symptom chosen from an increased osteoclast activity, increased bone resorption, bone lesion, hypercalcemia, a body weight loss, and any combinations thereof.
 12. The method of claim 10, wherein the tumor cell growth in bone includes bone cancer cells, and metastasized cancer cells originating from one or more of prostate cancer, breast cancer, lung cancer, thyroid cancer, renal cancer, ovarian cancer, pancreatic cancer, myeloma cancer, hepatoma, and melanoma.
 13. The method of claim 10, wherein said cancer comprises glioma.
 14. The method of claim 10, wherein said method further comprises administering to said mammal a therapeutically effective amount of one or more other pharmaceutical agents.
 15. The method of claim 14, wherein said other pharmaceutical agents are selected from the group consisting of an anti-cancer agent, an anti-inflammatory agent, an immune-modulating agent and an anti-osteoporosis agent.
 16. The method of claim 15, wherein said anti-cancer agent is selected from the group consisting of an antiangiogenic agent, a cytotoxic agent and an anti-neoplastic agent.
 17. The method of claim 16, wherein said method enhances the therapeutic efficacy of said anti-cancer agent.
 18. The method of claim 16, wherein said method allows administering to said patient a lower dose of said anti-cancer agent as compared to administering said anti-cancer agent without administering said isolated polypeptide, thereby decreasing side effects of said anti-cancer agent and/or decreasing resistance to said anti-cancer agent.
 19. The method of claim 16, wherein said method results in lowering the rate of cancer recurrence in said patient.
 20. The method of claim 10, wherein said cancer is associated with a highly expressed osteopontin.
 21. The method of claim 20, wherein said isolated polypeptide inhibits osteopontin-induced tumor invasion.
 22. An isolated polypeptide selective for αvβ3 integrin, wherein the polypeptide is a variant of a disintegrin which comprises a variant of SEQ ID NO: 29 that contains an amino acid substitution at position 3 and an optional substitution at one or more positions 1, 5 and 6 of SEQ ID NO:
 29. 23. The isolated polypeptide of claim 22, wherein said isolated polypeptide is pegylated or conjugated with albumin or conjugated with a tissue target molecule.
 24. The isolated polypeptide of claim 22, wherein the disintegrin is a variant of a disintegrin chosen from rhodostomin, albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, flavoridin, flavostatin, halysin, halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, and viridin.
 25. The isolated polypeptide of claim 24, wherein the disintegrin comprises a rhodostomin variant.
 26. The isolated polypeptide of claim 22, wherein said isolated polypeptide comprises an amino acid sequence chosen from SEQ ID NOs: 36-40.
 27. The isolated polypeptide of claim 22, wherein said isolated polypeptide comprises an amino acid sequence chosen from SEQ ID NOs: 63-67.
 28. The isolated polypeptide of claim 22, wherein said isolated polypeptide comprises SEQ ID NO:
 38. 29. The isolated polypeptide of claim 22, wherein said isolated polypeptide comprises SEQ ID NO:
 65. 30. A physiologically acceptable composition comprising the isolated polypeptide of claim 22, or a pharmaceutically acceptable salt of said polypeptide, and a pharmaceutically acceptable carrier.
 31. A physiologically acceptable composition comprising the isolated polypeptide of claim 26 and a pharmaceutically acceptable carrier.
 32. A physiologically acceptable composition comprising the isolated polypeptide of claim 27 and a pharmaceutically acceptable carrier.
 33. A physiologically acceptable composition comprising the isolated polypeptide of claim 28 and a pharmaceutically acceptable carrier.
 34. A physiologically acceptable composition comprising the isolated polypeptide of claim 29 and a pharmaceutically acceptable carrier.
 35. A method for making a polypeptide according to claim 22, comprising the steps of: a. transfecting a host cell with a polynucleotide encoding said polypeptide; b. growing said host cell in a culture medium; and c. isolating said polypeptide.
 36. The method of claim 35, wherein step (b) further comprises adding methanol to said culture medium.
 37. The method of claim 35, wherein said culture medium recited in step (b) is free of amino acids.
 38. The method of claim 35, wherein step (c) further comprises pegylating said polypeptide.
 39. The method of claim 35, wherein step (c) further comprises fusion of a tissue targeting molecule to the said polypeptide. 